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Electron Microscopy of Bone
CHAPTER
Electron Microscopy of Bone
4
V. Everts*,†, K. A. Hoeben† *Department of Oral Cell Biology, Academic Centre of Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands † Department of Cell Biology and Histology, Academic Medical Centre (AMC), University of Amsterdam, Amsterdam, The Netherlands
CHAPTER OUTLINE 1 Purpose����������������������������������������������������������������������������������������������������������������� 59 2 Theory������������������������������������������������������������������������������������������������������������������� 60 3 Equipments������������������������������������������������������������������������������������������������������������ 60 4 Materials��������������������������������������������������������������������������������������������������������������� 60 4.1 Solutions and Buffers—Step 1��������������������������������������������������������������� 60 4.2 Solutions and Buffers—Step 2��������������������������������������������������������������� 60 5 Protocol���������������������������������������������������������������������������������������������������������������� 61 5.1 Step 1—Preparation of the Solutions������������������������������������������������������ 61 5.2 Step 2—Fixation of Bone Samples��������������������������������������������������������� 63 5.3 Step 3—Decalcification of Bone Samples����������������������������������������������� 68 5.4 Step 4—Embedding of Bone Samples���������������������������������������������������� 68 5.5 Step 5—Sectioning of Bone Samples and Staining of the Sections������������ 75
Abstract Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultrastructural analysis. In general, the higher the level of mineralization is, the more difficult it is to obtain ultrathin sections of these tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem. In the present chapter we describe step-by-step how to prepare mineralized tissues for ultrastructural examination.
1 PURPOSE Description of the methods to visualize bone tissue at the ultra-structural level either with or without decalcification of the mineralized tissue. Methods in Cell Biology, Vol 113 Copyright © 2013 Elsevier Inc. All rights reserved.
0091-679X http://dx.doi.org/10.1016/B978-0-12-407239-8.00004-5
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CHAPTER 4 Electron Microscopy of Bone
2 THEORY Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultra-structural analysis. In general, the higher the level of mineralization, the more difficult obtaining the sections of non-decalcified tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem.
3 EQUIPMENTS Knife maker; ultramicrotome; transmission electron microscope; stoves for 40 and 60 °C.
4 MATERIALS Sodium cacodylate Glutardialdehyde Paraformaldehyde Osmium tetroxide Potassium ferrocyanide Uranyl acetate Lead nitrate Titriplex III (EDTA) Methylene blue Azure II Disodium tetratborat-10-hydrat Epoxy resin (LX-112, DDSA, NMA, DMP-30)
4.1 Solutions and Buffers—Step 1 Component
Final concentration
Sodium cacodylate buffer Glutaraldehyde Paraformaldehyde Osmium tetroxide
0.1 M 2.5% 1.0% 1.0%
Stock
Amount/L
25%
4.2 Solutions and Buffers—Step 2 Component
Final concentration
Sodium cacodylate
0.1 M
Stock
Amount/L
5 Protocol
5 PROTOCOL Duration
Time Preparation Protocol
Preparation Caution
About 1 day About 3–5 days or up to 3 weeks
Fix bone-tissue samples for electron microscopic evaluation The fixatives and buffers are hazardous solutions. Consult your safety officer
See Fig. 1 for the flowchart of the complete protocol.
5.1 Step 1—Preparation of the Solutions Overview Duration 1.1
Preparation of the fixative and buffer solutions About 1 day Fixative: 4% formaldehyde and 1% glutardialdehyde in 0.1 M sodium cacodylate buffer (pH 7.4).
Fix bone tissue samples for electron microscopic evaluation
Step 1 Preparation of the solutions
Step 2 Fixation of bone samples
Step 3 Decalcification of bone samples
Step 4 Embedding of bone samples
Step 5 Sectioning of bone samples and staining of the sections
FIGURE 1 Flowchart of the complete protocol including preparation.
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1.1.1 1.1.2 1.1.3 1.1.4 1.1.5 1.1.6 1.2
1.2.1
1.2.2
1.2.3 1.2.4 1.3 1.3.1 1.3.2
1.3.3 1.4
1.4.1 1.4.2 1.4.3
1.5
Heat 200 mL distilled water to 70 °C. Dissolve 40 g paraformaldehyde and add 2.5 g sodium hydroxide pellets. Allow the solution to cool (the solution should be clear). Add 21.4 g sodium cacodylate. Add 40 mL 25% glutardialdehyde and fill up to 800 mL with distilled water. Adjust the pH to 7.4 with 1 N HCl and fill up to 1000 mL. This solution should be stored at 4 °C and new fixative should be prepared each week. Osmium and ferrocyanide postfixative: 1% osmium tetroxide and 1.5% potassium ferrocyanide (K4Fe(CN)6)·3H2O) in 0.1 M sodium cacodylate buffer (pH 7.4). Stock solutions should be stored at 4 °C. 2% OsO4 stock solution: Add 1 g OsO4 crystals (crystalline, highest purity 99.95%) to 50 mL double distilled water in a stoppered dark-glass vial. Gently (!) shake the solution till the crystals are dissolved. Store the solution in the tightly closed vial at 4 °C. To avoid blackening of the solution, prior to its use, the vial has to be thoroughly cleaned with acetone to remove lipids (osmium is an excellent fixative for lipids!), washed in doubly distilled water and dried. Use gloves and avoid any contact with the skin! 0.2 M sodium cacodylate buffer: Dissolve 42.8 g sodium cacodylate in 900 mL distilled water. Adjust the pH to 7.4 and add distilled water to a volume of 1000 mL. 3% Ferrocyanide stock solution: Dissolve 3 g potassium ferrocyanide in 100 mL of 0.2 M sodium cacodylate buffer Prior to use, mix one volume of 2% OsO4 solution with one volume of 3% ferrocyanide solution. Osmium and cacodylate postfixative: 1% osmium tetroxide in 0.075 M sodium cacodylate buffer. 4% Osmium tetroxide stock solution: Dissolve 1 g OsO4 crystals in 25 mL distilled water according to the method described above. 0.1 M Sodium cacodylate buffer: Dissolve 21.4 g sodium cacodylate in 900 mL distilled water, adjust the pH to 7.4 and add distilled water to a volume of 1000 mL. Mix prior to fixation one volume of the 4% OsO4 solution with three volumes of 0.1 M sodium cacodylate buffer. Decalcification solution: 1.9% glutardialdehyde and 0.15 M EDTA (Titriplex III, ethylenedinitrilo tetraacetic acid disodium salt dihydrate) in 0.06 M sodium cacodylate buffer. Dissolve 38.53 g sodium cacodylate and 167.52 g Titriplex III in 2 L distilled water. Stir the solution and as soon as all Titriplex is dissolved (the solution should be clear) add 232 mL 25% glutardialdehyde. Adjust the pH to 7.4, first by adding approximately 10 g sodium hydroxide pellets followed by adding 2 N sodium hydroxide. Add distilled water to a volume of 3 L. This solution is stable for several months at 4 °C. Methylene blue
5 Protocol
1.5.1 1.5.2 1.5.3
Dissolve 2 g methylene blue in 100 mL distilled water (solution a) Dissolve 0.5 g Azure II in 50 mL distilled water (solution b). Dissolve 2 g Borax (di-sodium tetraborate-10-hydrate) in 100 mL distilled water (solution c). 1.5.4 Mix solutions a:b:c = 2:1:1, and store at 4 °C. Staining solution is stable for months. 1.5.5 Filter just before use. 1.6 Uranyl acetate 1.6.1 Dissolve 0.35 g uranyl acetate in 10 mL doubly distilled water. Store at 4 °C. 1.7 Lead nitrate 1.7.1 Boil and cool 50 mL doubly distilled water. 1.7.2 Dissolve 1.33 g lead nitrate and 1.76 g trisodium citrate dihydrate in 30 mL water. 1.7.3 Shake for 1 min vigorously and shake it a few times during a next 30 min. 1.7.4 Add 8 mL of 1 N NaOH and add cooled boiled-water to a volume of 50 mL. Store at 4 °C. 1.8 Epoxy resin Use gloves and a fume hood when preparing the stock of epoxy resin. 1.8.1 Mix under continuous stirring the resin components (Ladd Res. Industries, Burlington, Vermont), adding the next component when the previous one is completely dissolved. The components should be added in the following order: 100 g LX-112, 72.4 g DDSA, 40.4 g NMA and 3.9 g DMP-30. 1.8.2 Stir very well for another 30 min and collect the mixed resin in small plastic vials (10 mL) with cap. 1.8.3 Store these vials at −80 °C. The frozen vials can be kept at this temperature for a very long time (at least for 1 year). 1.8.4 Prior to embedding, warm an appropriate number of vials at ambient temperature. Open the vial only when the resin is at room temperature.
See Fig. 2 for the flowchart of Step 1.
5.2 Step 2—Fixation of Bone Samples Overview Duration 2.1 2.1.1
2.1.2 2.1.3
Fixation of tissue samples Between a few hours and 1 day Immersion fixation of animal bones After killing the animal and exposure of the bones of interest, dissect the bones and immerse them as quickly as possible in the fixative (freshly prepared 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4). If bones are collected from larger animals, the bones should be cut into smaller pieces. Cutting is preferably done in fixative (bones of young mice can be fixed without further cutting). Fix at ambient temperature for at least 4 h. After this the tissue samples can be left in fixative overnight at 4 °C. Wash the sample in 0.1 M sodium cacodylate buffer.
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Step 1 Preparation of the solutions Preparation of fixatives
1.1 Fixative: 4% formaldehyde and 1% glutardialdehyde in 0.1 M sodium cacodylate buffer (pH 7.4).
1.1.1 Heat 200 mL distilled water to 70 0C.
1.1.2 Dissolve 40 g paraformaldehyde and add 2.5 g sodium hydroxide pellets. Allow the solution to cool (the solution should be clear). 1.1.3 Add 21.4 g sodium cacodylate. 1.1.4 Add 40 ml 25% glutardialdehyde and fill up to 800 mL with distilled water 1.1.5 Adjust the pH to 7.4 with 1 N HCl and fill up to 1000 mL. 1.1.6 This solution should be stored at 4 oC and new fixative should be prepared each week.
1.2 Osmium and ferrocyanide post-fixative: 1% Osmium tetroxide and 1.5% potassium ferrocyanide (K4Fe(CN)6).3H2O) in 0.1 M sodium cacodylate buffer (pH 7.4). Stock solutions should be stored at 4 oC. 1.2.1 2% OsO4 stock solution: Add 1 g OsO4 crystals (Crystalline, highest purity 99.95%) to 50 ml double distilled water in a stoppered dark glass vial. Gently (!) shake the solution till the crystals are dissolved. Store the solution in the tightly closed vial at 4 0C. To avoid blackening of the solution, prior to its use the vial has to be thoroughly cleaned with acetone to remove lipids (osmium is an excellent fixative for lipids!), washed in doubly distilled water and dried. Use gloves and avoid any contact with the skin! 1.2.2 0.2 M sodium cacodylate buffer: Dissolve 42.8 g sodium cacodylate in 900 ml distilled water. Adjust the pH to 7.4 and add distilled water to a volume of 1000 ml.
1.2.3 3% Ferrocyanide stock solution: Dissolve 3 g potassium ferrocyanide in 100 ml 0.2 M sodium cacodylate buffer
1.2.4 Prior to use, mix one volume of 2% OsO4 solution with one volume of 3% ferrocyanide solution.
FIGURE 2 Flowchart of Step 1.
5 Protocol
1.3 Osmium and cacodylate post fixative: 1% Osmium tetroxide in 0.075 M sodium cacodylate buffer.
1.3.1 4% Osmium tetroxide stock solution: Dissolve 1 g OsO4 crystals in 25 ml distilled water according to the method described above.
1.3.2 0.1 M Sodium cacodylate buffer: Dissolve 21.4 g sodium cacodylate in 900 ml distilled water, adjust the pH to 7.4 and add distilled water to a volume of 1000 ml. 1.3.3 Mix prior to fixation one volume of the 4% OsO 4 solution with three volumes of 0.1 M sodium cacodylate buffer.
1.4. Decalcification solution: 1.9% glutardialdehyde and 0.15 M EDTA (Titriplex III, ethylenedinitrilo tetraacetic acid disodium salt dihydrate) in 0.06 M sodium cacodylate buffer.
1.4.1 Dissolve 38.53 g sodium cacodylate and 167.52 g Titriplex III in 2 l. distilled water
1.4.2 Stir the solution and as soon as all Titriplex is dissolved (the solution should be clear) add 232 ml 25% glutardialdehyde.
1.4.3 Adjust the pH to 7.4, first by adding approximately 10 g sodium hydroxide pellets followed by adding 2 N sodium hydroxide. Add distilled water to a volume of 3 l. This solution is stable for several months at 4 oC.
FIGURE 2—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
1.5. Methylene blue (staining solution for semi-thin sections)
1.5.1 Dissolve 2 g methylene blue in 100 ml distilled water (solution a)
1.5.2 Dissolve 0.5 g Azure II in 50 ml distilled water (solution b).
1.5.3 Dissolve 2 g Borax (di-sodiumtetraborat-10-hydrat) in 100 ml distilled water (solution c).
1.5.4 Mix solutions a:b:c= 2:1:1, and store at 4 oC. Staining solution is stable for months. Filter just before use.
1.6. Uranyl acetate
1.6.1 Dissolve 0.35 g uranyl acetate in 10 ml doubly distilled water. Store at 4 oC.
FIGURE 2—Cont’d
2.1.4 Transfer to postfixative for 1 h. 2.1.5 Wash the sample in doubly distilled water. 2.1.6 Proceed with the embedding protocol (Step 4). 2.2 Immersion fixation of human bone samples Immersion fixation and processing of bone samples obtained from humans is similar to protocol 2.2.1. It is essential that the samples are immersed into the fixative as quickly as possible and that the size of the fragments is small. Try to keep a maximal thickness of approximately 3–5 mm. Cutting of the bones into smaller fragments has to be performed in fixative.
1.7. Lead nitrate
1.7.1 Boil and cool 50 ml doubly distilled water.
1.7.2 Dissolve 1.33 g lead nitrate and 1.76 g tri-sodiumcitrate-dihydrate in 30 ml water.
1.7.3 Shake for 1 min vigorously and shake it a few times during a next 30 min.
1.7.4 Add 8 ml 1N NaOH and add cooled boiled water to a volume of 50 ml. Store at 4 oC.
1.8. Epoxy resin
Use gloves and a fume hood when preparing the stock of epoxy resin.
1.8.1 Mix under continuous stirring the resin components (Ladd Res. Industries, Burlington, Vermont), add the next component when the previous one is completely dissolved. The components should be added in the following order: 100 g LX-112, 72.4 g DDSA, 40.4 g NMA and 3.9 g DMP-30.
1.8.2 Stir very well for another 30 min and collect the mixed resin in small plastic vials (10 ml) with cap.
1.8.3 Store these vials at –800C. The frozen vials can be kept at this temperature for a very long time (at least for one year). 1.8.4 Prior to embedding, warm an appropriate number of vials at ambient temperature. Open the vial only when the resin is at room temperature.
FIGURE 2—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
2.3 2.3.1 2.3.2 2.3.3
Immersion fixation of cultured mineralized tissues Collect the bone explants after the preferred culture period and place in fixative. Leave at ambient temperature for at least 4 h (after this period the fixed bones can be kept in the fixative or in buffer at 4 °C for another day). Process the bones further with or without decalcification (see next part). Bones obtained from (very) young animals (e.g. mice < 10 days) can be processed without the decalcification step.
See Fig. 3 for the flowchart of Step 2.
5.3 Step 3—Decalcification of Bone Samples Overview Duration 3.1 3.1.1 3.1.2 3.1.3 3.1.4
Demineralization of calcified tissue samples About 2 to 3 weeks Decalcification of mineralized tissues Following fixation, immerse the bone samples in decalcification solution. Place the samples at 4 °C for 2 to 3 weeks, replacing the decalcification solution weekly. Check whether decalcification has been completed by X-ray photography. Once decalcification is complete, proceed to embedding.
See Fig. 4 for the flowchart of Step 3.
5.4 Step 4—Embedding of Bone Samples Embedding of mineralized or demineralized tissue samples About 3 to 4 days General approach to embedding and analysis of mineralized tissues by TEM Special care has to be taken in the embedding of calcified tissues since epoxy resins do not easily penetrate such tissues. Bones and/or teeth obtained from very young animals can be rather easily embedded in resin without giving problems with cutting and/or staining. Special attention has to be paid to embedding of bones from older and larger animals or man. 4.2 Embedding of small tissue samples Tissue samples obtained from young animals (up to 1 week old) of approximately 3–5 mm in thickness are put into glass (plastic dissolves in propylene oxide!) vials which can be closed and they are embedded as follows: 4.2.1 Immerse the sample in 70% ethanol for 3 × 5 min. 4.2.2 Replace with 80% ethanol for 3 × 5 min. 4.2.3 Replace with 90% ethanol for 3 × 5 min. 4.2.4 Replace with 96% ethanol for 3 × 5 min. 4.2.5 Immerse the tissue in 100% ethanol and close the vial. Incubate for 10 min. Repeat the 10 min incubation in 100% ethanol three times in total. 4.2.6 Replace the ethanol by propylene oxide. Incubate in a closed vial for 30 min. Change the propylene oxide and repeat this once. Overview Duration 4.1
5 Protocol
Step 2 Fixation of bone samples
2.1. Immersion fixation of animal bones
2.1.1 After killing the animal and exposure of the bones of interest, dissect the bones and immerse them as quickly as possible in the fixative (freshly prepared 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4). If bones are collected from larger animals, the bones should be cut into smaller pieces. Cutting is preferably done in fixative. (Bones of young small animals (e.g. mice) can be fixed without further cutting).
2.1.2 Fix at ambient temperature for at least 4 h. After this the tissue samples can be left in fixative overnight at 4 0 C.
2.1.3 Wash the sample in 0.1 M sodium cacodylate buffer.
2.1.4 Transfer to postfixative for 1 h.
2.1.5 Wash the sample in doubly distilled water.
2.1.6 Proceed with the embedding protocol (see Step 4).
FIGURE 3 Flowchart of Step 2.
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CHAPTER 4 Electron Microscopy of Bone
2.2. Immersion fixation of human bone samples
2.2.1 Immersion fixation and processing of bone samples obtained from humans is similar to Protocol 2.1. It is essential that the samples are immersed into the fixative as quickly as possible and that the size of the fragments is small. Try to keep a maximal thickness of approximately 3-5 mm. Cutting of the bones into smaller fragments has to be performed in fixative.
2.3. Immersion fixation of cultured mineralized tissues
2.3.1 Collect the bone explants after the preferred culture period and place them in fixative. 2.3.2 Leave at ambient temperature for at least 4h (after this period the fixed Bones can be kept in the fixative or in buffer at 4 0C for another day). 2.3.3 Process the bones further with or without decalcification (see next part). Bones obtained from (very) young animals (e.g. mice <10 days old) can be processed without the decalcification step.
FIGURE 3—Cont’d
4.2.7
4.2.8 4.2.9 4.2.10 4.3
Dissolve the epoxy resin in propylene oxide at a 1:1 concentration. Immerse the tissue samples in the epoxy resin-propylene mixture, close the vials and leave overnight while gentle shaking. Replace with pure epoxy resin and with the vials left open, place on a shaker and leave gently shaking for 5 h. Immerse the tissue samples in fresh epoxy resin in plastic molds, label and leave overnight in an oven at 40 °C. Transfer to an oven set at 60 °C to allow polymerization of the resin for 48 h. Embedding of larger bone samples
5 Protocol
Step 3 Decalcification of bone samples
3.1. Decalcification of mineralized tissues
3.1.1 Following fixation, immerse the bone samples in decalcification solution.
3.1.2 Place the samples at 4 0C for 2 to3 weeks, replacing the decalcification solution weekly.
3.1.3 Check whether decalcification has been completed by X-ray photography. Once decalcification is complete, proceed to embedding
FIGURE 4 Flowchart of Step 3.
It is possible to embed tissue samples of the size of a lower jaw of a full-grown mouse with good results. Larger samples have to be to be cut in smaller pieces in order to obtain sufficient penetration of the epoxy resin. 4.3.1 Follow the procedure as indicated under Subheading 4.2 but increase the duration of each ethanol dehydration step to 10 min and each propylene oxide impregnation step to 20 min. 4.3.2 Immerse the samples in propylene oxide:epoxy resin at a ratio of 3:1 for 3 h. 4.3.3 Immerse the samples in a 1:1 mixture of propylene oxide:epoxy resin for 3 h. 4.3.4 Immerse the samples in a 1:3 mixture of propylene oxide:epoxy resin overnight. 4.3.5 Immerse the samples in pure epoxy resin, with the vials open and shaking for 6 h. 4.3.6 Embed the samples in fresh epoxy resin and leave overnight in an oven at 40 °C, followed by polymerization at 60 °C during 48 h.
See Fig. 5 for the flowchart of Step 4.
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Step 4 Embedding of bone samples
4.1. General approach to embedding and analysis of mineralized tissues by TEM
4.1.1 Special care has to be taken in the embedding of calcified tissues since epoxy resins do not easily penetrate such tissues. Bones and/or teeth obtained from very young animals can be rather easily embedded in resin without giving problems with cutting and/or staining. Special attention has to be paid to embedding of bones from older and larger animals or man.
4.2. Embedding of small tissue samples
Tissue samples obtained from young animals (up to one week old) of approximately 3-5 mm in thickness are put into glass (plastic dissolves in propylene oxide!) vials which can be closed and they are embedded as follows:
4.2.1 Immerse the sample in 70% ethanol for 3 x 5 min.
4.2.2 Replace with 80% ethanol for 3 x 5 min 4.2.3 Replace with 90% ethanol for 3 x 5 min. 4.2.4 Replace with 96% ethanol for 3 x 5 min. 4.2.5 Immerse the tissue in 100% ethanol and close the vial. Incubate for 10 minutes. Repeat the 10 minutes incubation in 100% ethanol three times in total.
FIGURE 5 Flowchart of Step 4.
5 Protocol
4.2.6 Replace the ethanol by propylene oxide. Incubate in a closed vial for 30 minutes. Change the propylene oxide and repeat this once.
4.2.7 Dissolve the epoxy resin in propylene oxide at a 1:1 concentration. Immerse the tissue samples in the epoxy resin-propylene mixture, close the vials and leave overnight whilst gentle shaking.
4.2.8 Replace with pure epoxy resin and with the vials left open, place on a shaker and leave gently shaking for 5 h.
4.2.9 Immerse the tissue samples in fresh epoxy resin in plastic molds, label and leave overnight in an oven at 40 0C.
4.2.10 Transfer to an oven set at 60 0C to allow polymerization of the resin for 48 h.
FIGURE 5—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
4.3. Embedding of larger bone samples
It is possible to embed tissue samples of the size of a lower jaw of a full-grown mouse with good results. Larger samples have to be to be cut in smaller pieces in order to obtain sufficient penetration of the epoxy resin. 4.3.1 Follow the procedure as indicated under Subheading 4.2 but increase the duration of each ethanol dehydration step to 10 min and each propylene oxide impregnation step to 20 min. 4.3.2 Immerse the samples in propylene oxide:epoxy resin at a ratio of 3:1 for 3 h. 4.3.3 Immerse the samples in a 1:1 mixture of propylene oxide:epoxy resin for 3 h. 4.3.4 Immerse the samples in a 1:3 mixture of propylene oxide:epoxy resin overnight. 4.3.5 Immerse the samples in pure epoxy resin, with the vials open and shaking for 6 h. 4.3.6 Embed the samples in fresh epoxy resin and leave overnight in an oven at 40 0C, followed by polymerization at 60 0C during 48 h.
FIGURE 5—Con’d
5 Protocol
5.5 Step 5—Sectioning of Bone Samples and Staining of the Sections Overview Sectioning of plastic embedded tissue samples Duration A few hours 5.1 Sectioning mineralized tissues Sectioning of the tissue is best performed using diamond knives, which are available both for semi-thin and ultra-thin sectioning. 5.1.1 Use a glass knife to trim the block. 5.1.2 Proceed with semi-thin and ultra-thin sectioning using a diamond Histo-knife with the cutting angle set at 6°. 5.1.3 Make sure the microtome to manually cut at a very low speed (1 mm/s). 5.1.4 In order to avoid wetting of the surface of the tissue block, keep the water level of the trough as low as possible. 5.2 Methylene blue staining of semi-thin sections The methylene blue staining solution is an excellent stain for general purposes. Due to its metachromatic properties, some components (e.g. cartilage and granules of mast cells) stain purple. 5.2.1 Cut semi-thin sections of approximately 1–2 μm thickness. 5.2.2 Collect the sections on a drop of water on a glass slide and dry the sections on a heating plate (60–70 °C), leave the sections on the plate for an hour or longer. In order to avoid wrinkles of sections of larger tissue samples sections are best dried on a plate at 50 °C. Dry these sections overnight before staining. 5.2.3 When the sections are dry, add a drop of the filtered methylene blue staining solution to the section. 5.2.4 Leave the staining solution for approximately 15 s (depending on the type of tissue and thickness of the section). 5.2.5 Wash the section extensively with a jet of distilled water. 5.2.6 Dry the washed section and cover it with a drop of epoxy resin. 5.2.7 Cover the section with a cover glass and leave the section for several hours on a hot plate (or in a stove at 60 °C) to polymerize the resin. 5.3 Staining of ultrathin sections with uranyl acetate 5.3.1 Centrifuge the uranyl solution (10 min, 3000 rpm). 5.3.2 Put drops of uranyl acetate on a strip of parafilm. 5.3.3 Float the grids on top of the drops (sections facing the solution). 5.3.4 Stain the sections for 4–8 min in the dark. 5.3.5 Rinse the sections extensively with double distilled water. 5.3.6 Air-dry the sections and stain them with lead nitrate. 5.4 Staining of ultrathin sections with lead nitrate 5.4.1 Centrifuge the lead solution (10 min, 3000 g). 5.4.2 Put drops of lead nitrate on a strip of parafilm. 5.4.3 Place a few sodium hydroxide pellets around the drops. 5.4.4 Float the grids on top of the drops of the lead solution. 5.4.5 Cover the drops and pellets with a cover of a petri dish. 5.4.6 Stain the sections for 2–4 min. 5.4.7 Rinse the sections extensively with doubly distilled water. 5.4.8 Air-dry the sections.
See Fig. 6 for the flowchart of Step 5.
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Step 5 Sectioning of bone samples and staining of the sections
5.1. Sectioning mineralized tissues
Sectioning of the tissue is best performed using diamond knives, which are available both for semi-thin and ultrathin sectioning. 5.1.1 Use a glass knife to trim the block.
5.1.2 Proceed with semi-thin and ultra-thin sectioning using a diamond Histo-knife with the cutting angle set at 60.
5.2. Methylene blue staining of semi-thin sections
The methylene blue staining solution is an excellent stain for general purposes. Due to its metachromatic properties, some components (e.g. cartilage and granules of mast cells) stain purple.
5.2.1 Cut semi-thin sections of approximately 1to 2 µm thickness.
5.2.2 Collect the sections on a drop of water on a glass slide and dry the sections on a heating plate (60-70 0C), leave the sections on the plate for an hour or longer. In order to avoid wrinkles of sections of larger tissue samples sections are best dried on a plate at 50 0C. Dry these sections overnight before staining.
5.2.3 When the sections are dry, add a drop of the filtered methylene blue staining solution to the section.
FIGURE 6 Flowchart of Step 5.
5 Protocol
5.2.4 Leave the staining solution for approximately 15 sec (depending on the type of tissue and thickness of the section).
5.2.5 Wash the section extensively with a jet of distilled water.
5.2.6 Dry the washed section and cover it with a drop of epoxy resin. 5.2.7 Cover the section with a cover glass and leave the section for several hours on a hot plate (or in a stove at 60 0C) to polymerize the resin.
5.3. Staining of ultrathin sections with uranyl acetate
5.3.1 Centrifuge the uranyl solution (10 min, 3000 rpm).
5.3.2 Put drops of uranyl acetate on a strip of parafilm. 5.3.3 Float the grids on top of the drops (sections facing the solution). Stain the sections for 4-8 min in the dark. 5.3.4 Rinse the sections extensively with doubly distilled water. Air dry the sections and stain them with lead nitrate.
FIGURE 6—Cont’d
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5.4. Staining of ultrathin sections with lead nitrate
5.4.1 Centrifuge the lead solution (10 min, 3000 g).
5.4.2 Put drops of lead nitrate on a strip of parafilm.
5.4.3 Place a few sodium hydroxide pellets around the drops. Float the grids on top of the drops of the lead solution. 5.4.4 Cover the drops and pellets with a cover of a petri dish. Stain the sections for 2-4 min. 5.4.5 Rinse the sections extensively with doubly distilled water. Air dry the sections.
5.4.6 Examine the sections in the transmission electron microscope.
FIGURE 6—Cont’d
5 Protocol
Keywords Keyword Class Methods List the methods used to carry out this protocol (i.e. for each step). Process List the biological process(es) addressed in this protocol. Organisms List the primary organism used in this protocol. List any other applicable organisms. Pathways List any signaling, regulatory or metabolic pathways addressed in this protocol. Molecule Roles List any cellular or molecular roles addressed in this protocol. Molecule Functions List any cellular or molecular functions or activities addressed in this protocol. Phenotype List any developmental or functional phenotypes addressed in this protocol (organismal or cellular level). Anatomy List any gross anatomical structures, cellular structures, organelles or macromolecular complexes pertinent to this protocol. Diseases List any diseases or disease processes addressed in this protocol. Other List any other miscellaneous keywords that describe this protocol.
References Source article(s) used to create this protocol Everts, V., Niehof, A., Tigchelaar-Gutter, W., & Beertsen, W. (2012). Transmission electron microscopy of bone. Methods in Molecular Biology, 816, 351–363. PubMed PMID: 22130940.
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4
V. Everts*,†, K. A. Hoeben† *Department of Oral Cell Biology, Academic Centre of Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands † Department of Cell Biology and Histology, Academic Medical Centre (AMC), University of Amsterdam, Amsterdam, The Netherlands
CHAPTER OUTLINE 1 Purpose����������������������������������������������������������������������������������������������������������������� 59 2 Theory������������������������������������������������������������������������������������������������������������������� 60 3 Equipments������������������������������������������������������������������������������������������������������������ 60 4 Materials��������������������������������������������������������������������������������������������������������������� 60 4.1 Solutions and Buffers—Step 1��������������������������������������������������������������� 60 4.2 Solutions and Buffers—Step 2��������������������������������������������������������������� 60 5 Protocol���������������������������������������������������������������������������������������������������������������� 61 5.1 Step 1—Preparation of the Solutions������������������������������������������������������ 61 5.2 Step 2—Fixation of Bone Samples��������������������������������������������������������� 63 5.3 Step 3—Decalcification of Bone Samples����������������������������������������������� 68 5.4 Step 4—Embedding of Bone Samples���������������������������������������������������� 68 5.5 Step 5—Sectioning of Bone Samples and Staining of the Sections������������ 75
Abstract Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultrastructural analysis. In general, the higher the level of mineralization is, the more difficult it is to obtain ultrathin sections of these tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem. In the present chapter we describe step-by-step how to prepare mineralized tissues for ultrastructural examination.
1 PURPOSE Description of the methods to visualize bone tissue at the ultra-structural level either with or without decalcification of the mineralized tissue. Methods in Cell Biology, Vol 113 Copyright © 2013 Elsevier Inc. All rights reserved.
0091-679X http://dx.doi.org/10.1016/B978-0-12-407239-8.00004-5
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CHAPTER 4 Electron Microscopy of Bone
2 THEORY Mineralized tissues like bone, dentin and mineralized cartilage are difficult to prepare for ultra-structural analysis. In general, the higher the level of mineralization, the more difficult obtaining the sections of non-decalcified tissue. Tissues with a low level of mineral, e.g. from young animals, are rather simple to prepare and sectioning is not that much of a problem.
3 EQUIPMENTS Knife maker; ultramicrotome; transmission electron microscope; stoves for 40 and 60 °C.
4 MATERIALS Sodium cacodylate Glutardialdehyde Paraformaldehyde Osmium tetroxide Potassium ferrocyanide Uranyl acetate Lead nitrate Titriplex III (EDTA) Methylene blue Azure II Disodium tetratborat-10-hydrat Epoxy resin (LX-112, DDSA, NMA, DMP-30)
4.1 Solutions and Buffers—Step 1 Component
Final concentration
Sodium cacodylate buffer Glutaraldehyde Paraformaldehyde Osmium tetroxide
0.1 M 2.5% 1.0% 1.0%
Stock
Amount/L
25%
4.2 Solutions and Buffers—Step 2 Component
Final concentration
Sodium cacodylate
0.1 M
Stock
Amount/L
5 Protocol
5 PROTOCOL Duration
Time Preparation Protocol
Preparation Caution
About 1 day About 3–5 days or up to 3 weeks
Fix bone-tissue samples for electron microscopic evaluation The fixatives and buffers are hazardous solutions. Consult your safety officer
See Fig. 1 for the flowchart of the complete protocol.
5.1 Step 1—Preparation of the Solutions Overview Duration 1.1
Preparation of the fixative and buffer solutions About 1 day Fixative: 4% formaldehyde and 1% glutardialdehyde in 0.1 M sodium cacodylate buffer (pH 7.4).
Fix bone tissue samples for electron microscopic evaluation
Step 1 Preparation of the solutions
Step 2 Fixation of bone samples
Step 3 Decalcification of bone samples
Step 4 Embedding of bone samples
Step 5 Sectioning of bone samples and staining of the sections
FIGURE 1 Flowchart of the complete protocol including preparation.
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1.1.1 1.1.2 1.1.3 1.1.4 1.1.5 1.1.6 1.2
1.2.1
1.2.2
1.2.3 1.2.4 1.3 1.3.1 1.3.2
1.3.3 1.4
1.4.1 1.4.2 1.4.3
1.5
Heat 200 mL distilled water to 70 °C. Dissolve 40 g paraformaldehyde and add 2.5 g sodium hydroxide pellets. Allow the solution to cool (the solution should be clear). Add 21.4 g sodium cacodylate. Add 40 mL 25% glutardialdehyde and fill up to 800 mL with distilled water. Adjust the pH to 7.4 with 1 N HCl and fill up to 1000 mL. This solution should be stored at 4 °C and new fixative should be prepared each week. Osmium and ferrocyanide postfixative: 1% osmium tetroxide and 1.5% potassium ferrocyanide (K4Fe(CN)6)·3H2O) in 0.1 M sodium cacodylate buffer (pH 7.4). Stock solutions should be stored at 4 °C. 2% OsO4 stock solution: Add 1 g OsO4 crystals (crystalline, highest purity 99.95%) to 50 mL double distilled water in a stoppered dark-glass vial. Gently (!) shake the solution till the crystals are dissolved. Store the solution in the tightly closed vial at 4 °C. To avoid blackening of the solution, prior to its use, the vial has to be thoroughly cleaned with acetone to remove lipids (osmium is an excellent fixative for lipids!), washed in doubly distilled water and dried. Use gloves and avoid any contact with the skin! 0.2 M sodium cacodylate buffer: Dissolve 42.8 g sodium cacodylate in 900 mL distilled water. Adjust the pH to 7.4 and add distilled water to a volume of 1000 mL. 3% Ferrocyanide stock solution: Dissolve 3 g potassium ferrocyanide in 100 mL of 0.2 M sodium cacodylate buffer Prior to use, mix one volume of 2% OsO4 solution with one volume of 3% ferrocyanide solution. Osmium and cacodylate postfixative: 1% osmium tetroxide in 0.075 M sodium cacodylate buffer. 4% Osmium tetroxide stock solution: Dissolve 1 g OsO4 crystals in 25 mL distilled water according to the method described above. 0.1 M Sodium cacodylate buffer: Dissolve 21.4 g sodium cacodylate in 900 mL distilled water, adjust the pH to 7.4 and add distilled water to a volume of 1000 mL. Mix prior to fixation one volume of the 4% OsO4 solution with three volumes of 0.1 M sodium cacodylate buffer. Decalcification solution: 1.9% glutardialdehyde and 0.15 M EDTA (Titriplex III, ethylenedinitrilo tetraacetic acid disodium salt dihydrate) in 0.06 M sodium cacodylate buffer. Dissolve 38.53 g sodium cacodylate and 167.52 g Titriplex III in 2 L distilled water. Stir the solution and as soon as all Titriplex is dissolved (the solution should be clear) add 232 mL 25% glutardialdehyde. Adjust the pH to 7.4, first by adding approximately 10 g sodium hydroxide pellets followed by adding 2 N sodium hydroxide. Add distilled water to a volume of 3 L. This solution is stable for several months at 4 °C. Methylene blue
5 Protocol
1.5.1 1.5.2 1.5.3
Dissolve 2 g methylene blue in 100 mL distilled water (solution a) Dissolve 0.5 g Azure II in 50 mL distilled water (solution b). Dissolve 2 g Borax (di-sodium tetraborate-10-hydrate) in 100 mL distilled water (solution c). 1.5.4 Mix solutions a:b:c = 2:1:1, and store at 4 °C. Staining solution is stable for months. 1.5.5 Filter just before use. 1.6 Uranyl acetate 1.6.1 Dissolve 0.35 g uranyl acetate in 10 mL doubly distilled water. Store at 4 °C. 1.7 Lead nitrate 1.7.1 Boil and cool 50 mL doubly distilled water. 1.7.2 Dissolve 1.33 g lead nitrate and 1.76 g trisodium citrate dihydrate in 30 mL water. 1.7.3 Shake for 1 min vigorously and shake it a few times during a next 30 min. 1.7.4 Add 8 mL of 1 N NaOH and add cooled boiled-water to a volume of 50 mL. Store at 4 °C. 1.8 Epoxy resin Use gloves and a fume hood when preparing the stock of epoxy resin. 1.8.1 Mix under continuous stirring the resin components (Ladd Res. Industries, Burlington, Vermont), adding the next component when the previous one is completely dissolved. The components should be added in the following order: 100 g LX-112, 72.4 g DDSA, 40.4 g NMA and 3.9 g DMP-30. 1.8.2 Stir very well for another 30 min and collect the mixed resin in small plastic vials (10 mL) with cap. 1.8.3 Store these vials at −80 °C. The frozen vials can be kept at this temperature for a very long time (at least for 1 year). 1.8.4 Prior to embedding, warm an appropriate number of vials at ambient temperature. Open the vial only when the resin is at room temperature.
See Fig. 2 for the flowchart of Step 1.
5.2 Step 2—Fixation of Bone Samples Overview Duration 2.1 2.1.1
2.1.2 2.1.3
Fixation of tissue samples Between a few hours and 1 day Immersion fixation of animal bones After killing the animal and exposure of the bones of interest, dissect the bones and immerse them as quickly as possible in the fixative (freshly prepared 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4). If bones are collected from larger animals, the bones should be cut into smaller pieces. Cutting is preferably done in fixative (bones of young mice can be fixed without further cutting). Fix at ambient temperature for at least 4 h. After this the tissue samples can be left in fixative overnight at 4 °C. Wash the sample in 0.1 M sodium cacodylate buffer.
63
Step 1 Preparation of the solutions Preparation of fixatives
1.1 Fixative: 4% formaldehyde and 1% glutardialdehyde in 0.1 M sodium cacodylate buffer (pH 7.4).
1.1.1 Heat 200 mL distilled water to 70 0C.
1.1.2 Dissolve 40 g paraformaldehyde and add 2.5 g sodium hydroxide pellets. Allow the solution to cool (the solution should be clear). 1.1.3 Add 21.4 g sodium cacodylate. 1.1.4 Add 40 ml 25% glutardialdehyde and fill up to 800 mL with distilled water 1.1.5 Adjust the pH to 7.4 with 1 N HCl and fill up to 1000 mL. 1.1.6 This solution should be stored at 4 oC and new fixative should be prepared each week.
1.2 Osmium and ferrocyanide post-fixative: 1% Osmium tetroxide and 1.5% potassium ferrocyanide (K4Fe(CN)6).3H2O) in 0.1 M sodium cacodylate buffer (pH 7.4). Stock solutions should be stored at 4 oC. 1.2.1 2% OsO4 stock solution: Add 1 g OsO4 crystals (Crystalline, highest purity 99.95%) to 50 ml double distilled water in a stoppered dark glass vial. Gently (!) shake the solution till the crystals are dissolved. Store the solution in the tightly closed vial at 4 0C. To avoid blackening of the solution, prior to its use the vial has to be thoroughly cleaned with acetone to remove lipids (osmium is an excellent fixative for lipids!), washed in doubly distilled water and dried. Use gloves and avoid any contact with the skin! 1.2.2 0.2 M sodium cacodylate buffer: Dissolve 42.8 g sodium cacodylate in 900 ml distilled water. Adjust the pH to 7.4 and add distilled water to a volume of 1000 ml.
1.2.3 3% Ferrocyanide stock solution: Dissolve 3 g potassium ferrocyanide in 100 ml 0.2 M sodium cacodylate buffer
1.2.4 Prior to use, mix one volume of 2% OsO4 solution with one volume of 3% ferrocyanide solution.
FIGURE 2 Flowchart of Step 1.
5 Protocol
1.3 Osmium and cacodylate post fixative: 1% Osmium tetroxide in 0.075 M sodium cacodylate buffer.
1.3.1 4% Osmium tetroxide stock solution: Dissolve 1 g OsO4 crystals in 25 ml distilled water according to the method described above.
1.3.2 0.1 M Sodium cacodylate buffer: Dissolve 21.4 g sodium cacodylate in 900 ml distilled water, adjust the pH to 7.4 and add distilled water to a volume of 1000 ml. 1.3.3 Mix prior to fixation one volume of the 4% OsO 4 solution with three volumes of 0.1 M sodium cacodylate buffer.
1.4. Decalcification solution: 1.9% glutardialdehyde and 0.15 M EDTA (Titriplex III, ethylenedinitrilo tetraacetic acid disodium salt dihydrate) in 0.06 M sodium cacodylate buffer.
1.4.1 Dissolve 38.53 g sodium cacodylate and 167.52 g Titriplex III in 2 l. distilled water
1.4.2 Stir the solution and as soon as all Titriplex is dissolved (the solution should be clear) add 232 ml 25% glutardialdehyde.
1.4.3 Adjust the pH to 7.4, first by adding approximately 10 g sodium hydroxide pellets followed by adding 2 N sodium hydroxide. Add distilled water to a volume of 3 l. This solution is stable for several months at 4 oC.
FIGURE 2—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
1.5. Methylene blue (staining solution for semi-thin sections)
1.5.1 Dissolve 2 g methylene blue in 100 ml distilled water (solution a)
1.5.2 Dissolve 0.5 g Azure II in 50 ml distilled water (solution b).
1.5.3 Dissolve 2 g Borax (di-sodiumtetraborat-10-hydrat) in 100 ml distilled water (solution c).
1.5.4 Mix solutions a:b:c= 2:1:1, and store at 4 oC. Staining solution is stable for months. Filter just before use.
1.6. Uranyl acetate
1.6.1 Dissolve 0.35 g uranyl acetate in 10 ml doubly distilled water. Store at 4 oC.
FIGURE 2—Cont’d
2.1.4 Transfer to postfixative for 1 h. 2.1.5 Wash the sample in doubly distilled water. 2.1.6 Proceed with the embedding protocol (Step 4). 2.2 Immersion fixation of human bone samples Immersion fixation and processing of bone samples obtained from humans is similar to protocol 2.2.1. It is essential that the samples are immersed into the fixative as quickly as possible and that the size of the fragments is small. Try to keep a maximal thickness of approximately 3–5 mm. Cutting of the bones into smaller fragments has to be performed in fixative.
1.7. Lead nitrate
1.7.1 Boil and cool 50 ml doubly distilled water.
1.7.2 Dissolve 1.33 g lead nitrate and 1.76 g tri-sodiumcitrate-dihydrate in 30 ml water.
1.7.3 Shake for 1 min vigorously and shake it a few times during a next 30 min.
1.7.4 Add 8 ml 1N NaOH and add cooled boiled water to a volume of 50 ml. Store at 4 oC.
1.8. Epoxy resin
Use gloves and a fume hood when preparing the stock of epoxy resin.
1.8.1 Mix under continuous stirring the resin components (Ladd Res. Industries, Burlington, Vermont), add the next component when the previous one is completely dissolved. The components should be added in the following order: 100 g LX-112, 72.4 g DDSA, 40.4 g NMA and 3.9 g DMP-30.
1.8.2 Stir very well for another 30 min and collect the mixed resin in small plastic vials (10 ml) with cap.
1.8.3 Store these vials at –800C. The frozen vials can be kept at this temperature for a very long time (at least for one year). 1.8.4 Prior to embedding, warm an appropriate number of vials at ambient temperature. Open the vial only when the resin is at room temperature.
FIGURE 2—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
2.3 2.3.1 2.3.2 2.3.3
Immersion fixation of cultured mineralized tissues Collect the bone explants after the preferred culture period and place in fixative. Leave at ambient temperature for at least 4 h (after this period the fixed bones can be kept in the fixative or in buffer at 4 °C for another day). Process the bones further with or without decalcification (see next part). Bones obtained from (very) young animals (e.g. mice < 10 days) can be processed without the decalcification step.
See Fig. 3 for the flowchart of Step 2.
5.3 Step 3—Decalcification of Bone Samples Overview Duration 3.1 3.1.1 3.1.2 3.1.3 3.1.4
Demineralization of calcified tissue samples About 2 to 3 weeks Decalcification of mineralized tissues Following fixation, immerse the bone samples in decalcification solution. Place the samples at 4 °C for 2 to 3 weeks, replacing the decalcification solution weekly. Check whether decalcification has been completed by X-ray photography. Once decalcification is complete, proceed to embedding.
See Fig. 4 for the flowchart of Step 3.
5.4 Step 4—Embedding of Bone Samples Embedding of mineralized or demineralized tissue samples About 3 to 4 days General approach to embedding and analysis of mineralized tissues by TEM Special care has to be taken in the embedding of calcified tissues since epoxy resins do not easily penetrate such tissues. Bones and/or teeth obtained from very young animals can be rather easily embedded in resin without giving problems with cutting and/or staining. Special attention has to be paid to embedding of bones from older and larger animals or man. 4.2 Embedding of small tissue samples Tissue samples obtained from young animals (up to 1 week old) of approximately 3–5 mm in thickness are put into glass (plastic dissolves in propylene oxide!) vials which can be closed and they are embedded as follows: 4.2.1 Immerse the sample in 70% ethanol for 3 × 5 min. 4.2.2 Replace with 80% ethanol for 3 × 5 min. 4.2.3 Replace with 90% ethanol for 3 × 5 min. 4.2.4 Replace with 96% ethanol for 3 × 5 min. 4.2.5 Immerse the tissue in 100% ethanol and close the vial. Incubate for 10 min. Repeat the 10 min incubation in 100% ethanol three times in total. 4.2.6 Replace the ethanol by propylene oxide. Incubate in a closed vial for 30 min. Change the propylene oxide and repeat this once. Overview Duration 4.1
5 Protocol
Step 2 Fixation of bone samples
2.1. Immersion fixation of animal bones
2.1.1 After killing the animal and exposure of the bones of interest, dissect the bones and immerse them as quickly as possible in the fixative (freshly prepared 4% formaldehyde and 1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4). If bones are collected from larger animals, the bones should be cut into smaller pieces. Cutting is preferably done in fixative. (Bones of young small animals (e.g. mice) can be fixed without further cutting).
2.1.2 Fix at ambient temperature for at least 4 h. After this the tissue samples can be left in fixative overnight at 4 0 C.
2.1.3 Wash the sample in 0.1 M sodium cacodylate buffer.
2.1.4 Transfer to postfixative for 1 h.
2.1.5 Wash the sample in doubly distilled water.
2.1.6 Proceed with the embedding protocol (see Step 4).
FIGURE 3 Flowchart of Step 2.
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CHAPTER 4 Electron Microscopy of Bone
2.2. Immersion fixation of human bone samples
2.2.1 Immersion fixation and processing of bone samples obtained from humans is similar to Protocol 2.1. It is essential that the samples are immersed into the fixative as quickly as possible and that the size of the fragments is small. Try to keep a maximal thickness of approximately 3-5 mm. Cutting of the bones into smaller fragments has to be performed in fixative.
2.3. Immersion fixation of cultured mineralized tissues
2.3.1 Collect the bone explants after the preferred culture period and place them in fixative. 2.3.2 Leave at ambient temperature for at least 4h (after this period the fixed Bones can be kept in the fixative or in buffer at 4 0C for another day). 2.3.3 Process the bones further with or without decalcification (see next part). Bones obtained from (very) young animals (e.g. mice <10 days old) can be processed without the decalcification step.
FIGURE 3—Cont’d
4.2.7
4.2.8 4.2.9 4.2.10 4.3
Dissolve the epoxy resin in propylene oxide at a 1:1 concentration. Immerse the tissue samples in the epoxy resin-propylene mixture, close the vials and leave overnight while gentle shaking. Replace with pure epoxy resin and with the vials left open, place on a shaker and leave gently shaking for 5 h. Immerse the tissue samples in fresh epoxy resin in plastic molds, label and leave overnight in an oven at 40 °C. Transfer to an oven set at 60 °C to allow polymerization of the resin for 48 h. Embedding of larger bone samples
5 Protocol
Step 3 Decalcification of bone samples
3.1. Decalcification of mineralized tissues
3.1.1 Following fixation, immerse the bone samples in decalcification solution.
3.1.2 Place the samples at 4 0C for 2 to3 weeks, replacing the decalcification solution weekly.
3.1.3 Check whether decalcification has been completed by X-ray photography. Once decalcification is complete, proceed to embedding
FIGURE 4 Flowchart of Step 3.
It is possible to embed tissue samples of the size of a lower jaw of a full-grown mouse with good results. Larger samples have to be to be cut in smaller pieces in order to obtain sufficient penetration of the epoxy resin. 4.3.1 Follow the procedure as indicated under Subheading 4.2 but increase the duration of each ethanol dehydration step to 10 min and each propylene oxide impregnation step to 20 min. 4.3.2 Immerse the samples in propylene oxide:epoxy resin at a ratio of 3:1 for 3 h. 4.3.3 Immerse the samples in a 1:1 mixture of propylene oxide:epoxy resin for 3 h. 4.3.4 Immerse the samples in a 1:3 mixture of propylene oxide:epoxy resin overnight. 4.3.5 Immerse the samples in pure epoxy resin, with the vials open and shaking for 6 h. 4.3.6 Embed the samples in fresh epoxy resin and leave overnight in an oven at 40 °C, followed by polymerization at 60 °C during 48 h.
See Fig. 5 for the flowchart of Step 4.
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CHAPTER 4 Electron Microscopy of Bone
Step 4 Embedding of bone samples
4.1. General approach to embedding and analysis of mineralized tissues by TEM
4.1.1 Special care has to be taken in the embedding of calcified tissues since epoxy resins do not easily penetrate such tissues. Bones and/or teeth obtained from very young animals can be rather easily embedded in resin without giving problems with cutting and/or staining. Special attention has to be paid to embedding of bones from older and larger animals or man.
4.2. Embedding of small tissue samples
Tissue samples obtained from young animals (up to one week old) of approximately 3-5 mm in thickness are put into glass (plastic dissolves in propylene oxide!) vials which can be closed and they are embedded as follows:
4.2.1 Immerse the sample in 70% ethanol for 3 x 5 min.
4.2.2 Replace with 80% ethanol for 3 x 5 min 4.2.3 Replace with 90% ethanol for 3 x 5 min. 4.2.4 Replace with 96% ethanol for 3 x 5 min. 4.2.5 Immerse the tissue in 100% ethanol and close the vial. Incubate for 10 minutes. Repeat the 10 minutes incubation in 100% ethanol three times in total.
FIGURE 5 Flowchart of Step 4.
5 Protocol
4.2.6 Replace the ethanol by propylene oxide. Incubate in a closed vial for 30 minutes. Change the propylene oxide and repeat this once.
4.2.7 Dissolve the epoxy resin in propylene oxide at a 1:1 concentration. Immerse the tissue samples in the epoxy resin-propylene mixture, close the vials and leave overnight whilst gentle shaking.
4.2.8 Replace with pure epoxy resin and with the vials left open, place on a shaker and leave gently shaking for 5 h.
4.2.9 Immerse the tissue samples in fresh epoxy resin in plastic molds, label and leave overnight in an oven at 40 0C.
4.2.10 Transfer to an oven set at 60 0C to allow polymerization of the resin for 48 h.
FIGURE 5—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
4.3. Embedding of larger bone samples
It is possible to embed tissue samples of the size of a lower jaw of a full-grown mouse with good results. Larger samples have to be to be cut in smaller pieces in order to obtain sufficient penetration of the epoxy resin. 4.3.1 Follow the procedure as indicated under Subheading 4.2 but increase the duration of each ethanol dehydration step to 10 min and each propylene oxide impregnation step to 20 min. 4.3.2 Immerse the samples in propylene oxide:epoxy resin at a ratio of 3:1 for 3 h. 4.3.3 Immerse the samples in a 1:1 mixture of propylene oxide:epoxy resin for 3 h. 4.3.4 Immerse the samples in a 1:3 mixture of propylene oxide:epoxy resin overnight. 4.3.5 Immerse the samples in pure epoxy resin, with the vials open and shaking for 6 h. 4.3.6 Embed the samples in fresh epoxy resin and leave overnight in an oven at 40 0C, followed by polymerization at 60 0C during 48 h.
FIGURE 5—Con’d
5 Protocol
5.5 Step 5—Sectioning of Bone Samples and Staining of the Sections Overview Sectioning of plastic embedded tissue samples Duration A few hours 5.1 Sectioning mineralized tissues Sectioning of the tissue is best performed using diamond knives, which are available both for semi-thin and ultra-thin sectioning. 5.1.1 Use a glass knife to trim the block. 5.1.2 Proceed with semi-thin and ultra-thin sectioning using a diamond Histo-knife with the cutting angle set at 6°. 5.1.3 Make sure the microtome to manually cut at a very low speed (1 mm/s). 5.1.4 In order to avoid wetting of the surface of the tissue block, keep the water level of the trough as low as possible. 5.2 Methylene blue staining of semi-thin sections The methylene blue staining solution is an excellent stain for general purposes. Due to its metachromatic properties, some components (e.g. cartilage and granules of mast cells) stain purple. 5.2.1 Cut semi-thin sections of approximately 1–2 μm thickness. 5.2.2 Collect the sections on a drop of water on a glass slide and dry the sections on a heating plate (60–70 °C), leave the sections on the plate for an hour or longer. In order to avoid wrinkles of sections of larger tissue samples sections are best dried on a plate at 50 °C. Dry these sections overnight before staining. 5.2.3 When the sections are dry, add a drop of the filtered methylene blue staining solution to the section. 5.2.4 Leave the staining solution for approximately 15 s (depending on the type of tissue and thickness of the section). 5.2.5 Wash the section extensively with a jet of distilled water. 5.2.6 Dry the washed section and cover it with a drop of epoxy resin. 5.2.7 Cover the section with a cover glass and leave the section for several hours on a hot plate (or in a stove at 60 °C) to polymerize the resin. 5.3 Staining of ultrathin sections with uranyl acetate 5.3.1 Centrifuge the uranyl solution (10 min, 3000 rpm). 5.3.2 Put drops of uranyl acetate on a strip of parafilm. 5.3.3 Float the grids on top of the drops (sections facing the solution). 5.3.4 Stain the sections for 4–8 min in the dark. 5.3.5 Rinse the sections extensively with double distilled water. 5.3.6 Air-dry the sections and stain them with lead nitrate. 5.4 Staining of ultrathin sections with lead nitrate 5.4.1 Centrifuge the lead solution (10 min, 3000 g). 5.4.2 Put drops of lead nitrate on a strip of parafilm. 5.4.3 Place a few sodium hydroxide pellets around the drops. 5.4.4 Float the grids on top of the drops of the lead solution. 5.4.5 Cover the drops and pellets with a cover of a petri dish. 5.4.6 Stain the sections for 2–4 min. 5.4.7 Rinse the sections extensively with doubly distilled water. 5.4.8 Air-dry the sections.
See Fig. 6 for the flowchart of Step 5.
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Step 5 Sectioning of bone samples and staining of the sections
5.1. Sectioning mineralized tissues
Sectioning of the tissue is best performed using diamond knives, which are available both for semi-thin and ultrathin sectioning. 5.1.1 Use a glass knife to trim the block.
5.1.2 Proceed with semi-thin and ultra-thin sectioning using a diamond Histo-knife with the cutting angle set at 60.
5.2. Methylene blue staining of semi-thin sections
The methylene blue staining solution is an excellent stain for general purposes. Due to its metachromatic properties, some components (e.g. cartilage and granules of mast cells) stain purple.
5.2.1 Cut semi-thin sections of approximately 1to 2 µm thickness.
5.2.2 Collect the sections on a drop of water on a glass slide and dry the sections on a heating plate (60-70 0C), leave the sections on the plate for an hour or longer. In order to avoid wrinkles of sections of larger tissue samples sections are best dried on a plate at 50 0C. Dry these sections overnight before staining.
5.2.3 When the sections are dry, add a drop of the filtered methylene blue staining solution to the section.
FIGURE 6 Flowchart of Step 5.
5 Protocol
5.2.4 Leave the staining solution for approximately 15 sec (depending on the type of tissue and thickness of the section).
5.2.5 Wash the section extensively with a jet of distilled water.
5.2.6 Dry the washed section and cover it with a drop of epoxy resin. 5.2.7 Cover the section with a cover glass and leave the section for several hours on a hot plate (or in a stove at 60 0C) to polymerize the resin.
5.3. Staining of ultrathin sections with uranyl acetate
5.3.1 Centrifuge the uranyl solution (10 min, 3000 rpm).
5.3.2 Put drops of uranyl acetate on a strip of parafilm. 5.3.3 Float the grids on top of the drops (sections facing the solution). Stain the sections for 4-8 min in the dark. 5.3.4 Rinse the sections extensively with doubly distilled water. Air dry the sections and stain them with lead nitrate.
FIGURE 6—Cont’d
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CHAPTER 4 Electron Microscopy of Bone
5.4. Staining of ultrathin sections with lead nitrate
5.4.1 Centrifuge the lead solution (10 min, 3000 g).
5.4.2 Put drops of lead nitrate on a strip of parafilm.
5.4.3 Place a few sodium hydroxide pellets around the drops. Float the grids on top of the drops of the lead solution. 5.4.4 Cover the drops and pellets with a cover of a petri dish. Stain the sections for 2-4 min. 5.4.5 Rinse the sections extensively with doubly distilled water. Air dry the sections.
5.4.6 Examine the sections in the transmission electron microscope.
FIGURE 6—Cont’d
5 Protocol
Keywords Keyword Class Methods List the methods used to carry out this protocol (i.e. for each step). Process List the biological process(es) addressed in this protocol. Organisms List the primary organism used in this protocol. List any other applicable organisms. Pathways List any signaling, regulatory or metabolic pathways addressed in this protocol. Molecule Roles List any cellular or molecular roles addressed in this protocol. Molecule Functions List any cellular or molecular functions or activities addressed in this protocol. Phenotype List any developmental or functional phenotypes addressed in this protocol (organismal or cellular level). Anatomy List any gross anatomical structures, cellular structures, organelles or macromolecular complexes pertinent to this protocol. Diseases List any diseases or disease processes addressed in this protocol. Other List any other miscellaneous keywords that describe this protocol.
References Source article(s) used to create this protocol Everts, V., Niehof, A., Tigchelaar-Gutter, W., & Beertsen, W. (2012). Transmission electron microscopy of bone. Methods in Molecular Biology, 816, 351–363. PubMed PMID: 22130940.
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