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An efficient standardized method of staining thin sections for electron microscopy
Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved
180
J. ULTRASTRUCTURE RESEARCH
42, 180-185 (1973)
An Efficient Standardized Method of Staining Thin Sections for Electron Microscopy M. SJOSTROM,L.-E. THORNELL,and S. HELLSTROM
Department of Anatomy, University of Umed, S-901 87 Umed, Sweden Received June 20, 1972 A simple, rapid, and reproducible method is described for staining of thin sections for electron microscopy. A modified specimen grid box (LKB) is used which holds 100 grids. The sliding cover as well as the bottoms of the compartments of the grid box are bored through. The box is placed in a trough containing the staining solution and then rinsed in beakers with water. The simple and still most widely applied method of staining thin sections for electron microscopy, as described in handbooks on techniques for electron microscopy (6, 8, 14), was introduced by Gibbons and Bradfield in 1956 (4) and was later presented in greater detail by Watson in 1958 (15). The thin sections are collected on a grid, coated with a supporting film or uncoated. Thereafter the grid is either floated on the surface of the staining solution with the section side down or it is immersed in the staining solution. After staining for the necessary time, the sections are immediately rinsed by holding the grid with a forceps and gently flushing the surface of the sections with distilled water. The grid is then placed to dry. This method of staining thin sections has, however, certain disadvantages. The staining solution is to a large extent in contact with air which frequently leads to formation of an insoluble stain precipitate; this in turn may contaminate the sections, e.g., when removing the grids from the stain droplets prior to rinsing process or if some of the staining solution is trapped on the grid or in the forceps when the grid is placed to dry. If many grids are to be stained, the process is time-consuming, especially if multiple sequential staining is performed. Also, reproducibility is rendered more difficult when the staining time, especially if short, is not precisely the same for all grids. Furthermore, the supporting film may be damaged by the repeated use of forceps. This technical communication describes a method of staining up to 100 grids simultaneously. The method also eliminates many sources of artifacts due to precipitated stain, disrupted supporting films, and damaged sections or to varying staining and rinsing times.
STANDARDIZEDSTAINING OF THIN SECTIONS
181
MATERIALS The method is based on the use of a modified specimen grid box, LKB-produkter AB, S-161 25 Bromma, Sweden, No. 4828B, which holds 100 grids in 20 rows of 5 diamondshaped holes (Fig. 1). The sliding cover as well as the bottoms of the plastic box are bored through (Fig. 2). The diameter of the bore-holes is equal to the shortest distance between the parallel edges of the diamond-shaped compartments. A rod-shaped handle, about 40 mm in length, is mounted on the cover. A flat trough just large enough to accommodate the grid box and supplied with a lid is also required (Figs. 3 and 4).
STAINING PROCEDURE W a s h the modified specimen grid box and the trough before use. H o l d the grid box by the handle to avoid contamination by fingerprints. 1. Transfer the grids f r o m the ordinary grid box, where they have been stored since the sectioning procedure, to the staining grid box (Fig. 1). 2. Pour the staining solution into the t r o u g h just prior to the c o m m e n c e m e n t of the staining procedure. 3. Place the box in the t r o u g h (Fig. 3). The staining solution rapidly fills the compartments containing the grids. Cover the t r o u g h with the lid as quickly as possible. 4. After a suitable staining time, remove the lid, and transfer the box to a beaker containing fresh distilled water. Agitate carefully for 5-10 seconds (Fig. 4). 5. Pour rinsing fluid over the end of the tilted box to remove any staining solution that m a y be trapped between the cover and the plastic box (Fig. 5). 6. Finally, wash the box in a second beaker of rinsing solution (Fig. 6). The grid container m a y be dried in several ways, e.g., by placing it on end on a filter paper or some other absorbent material. If multiple sequential staining is desired, simply wash the t r o u g h thoroughly or, preferably, use another one, change the fluids in the beakers, and repeat the steps described above.
FIG. 1. A normal specimen grid box (to the right) compared with the modified grid box (to the left) in which both the sliding cover and the bottoms of the compartments of the plastic box have been bored through. A handle is mounted on the cover. The grids are transferred from the compartments of the ordinary grid box to the corresponding compartments of the staining box. FIG. 2. Schematic drawing showing the grid placed in the diamond-shaped compartment. FIG. 3. The staining grid box has been placed in the trough containing the staining solution which now fills the compartments of the box. Note that the lid (see Fig. 4) is missing for the sake of clarity. Normally the lid covers the trough throughout the staining period. FIG. 4. The box is washed by carefully moving it up and down in a beaker filled with distilled water. FIG. 5. Any staining solution which may be lying between the cover and the plastic box is eliminated by pouring water over the end of the box. FIG. 6. Finally the box is washed in a second beaker of water.
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STANDARDIZED STAINING OF THIN SECTIONS
183
184
SJOSTROM, THORNELL,AND HELLSTR(JlV[
DISCUSSION Many attempts have been made to automate and standardize the different steps in the processing of tissues for electron microscopy (1, 10). These have mostly involved technically advanced and expensive apparatus. Several methods of staining sections mounted on grids other than those discussed in the introduction are in use [cf. Hayat (6)]. Only a few authors, however, have tried to improve the techniques of staining, rinsing, and drying. Gorycki (5) introduced a device which makes it possible to stain 10 grids simultaneously. Feldman (3) presented an apparatus consisting of a series of wells filled with staining solution, at the bottoms of which the grids were placed. However, neither these nor other devices (2, 7, 9, 11-13) seem to be widely adopted, probably because they are not commercially available. Our method of staining thin sections is time-saving and simple to apply. It standardizes the staining; all sections are stained and rinsed for exactly the same time period. This is of advantage when studying serial sections and when comparing the density of structures. The protection provided by the grid box reduces the risk of mechanically ruining sections and supporting films. Any number of successive stains and rinses can be applied without removing the grids from the box. Furthermore, only small amounts (a few milliliters) of stain are required, and the chances of intermingling the grids are minimized. The surface tension of the solutions maintains complete filling of the compartments of the grid box when the box is lifted out of the solutions. The grids are therefore surrounded by staining solution until the moment when the box is immersed in the water rinse and therefore they do not encounter a liquid-air interface at which contaminants may accumulate [see discussion by, e.g., Dougherty (2) and Rowden (13)]. No deposits of precipitated stain are found on the sections, which is perhaps also due to the small area of contact between air and stain. Naturally, the risk of contamination of the sections, by sedimentation of precipitate formed at the surface, is not entirely eliminated. There are, however, other ways to deal with this problem, should it arise. Normann (9) recommended placing grids in envelopes made of filter paper. Similarly, a piece of filter paper may be placed on the cover in contact with the staining solution. The filter paper will then retain any precipitate that may form. If staining solutions which are extremely sensitive to air influence are employed, it is possible to place the grid box and the trough in a suitable chamber gassed with, e.g., nitrogen. The technique of staining thin sections described in this paper has been tested, compared with the standard method, and applied at our laboratory for about one year. The results are fully comparable to carefully performed routine staining of single grids. Naturally, great care must still be exercised when preparing the staining solutions and cleaning glassware and other accessories.
STANDARDIZED STAININGOF THIN SECTIONS
185
This project was supported by grants from the Medical Faculty of Ume~t and from C.-B. Nathhorsts vetenskapliga stiftelse. REFERENCES 1. AIHARA,K., N o ~ z o , K., NAGATA, K., NISm~ZAWA, H. and Suzu~a, K., J. Electronmicrosc. 16, 285 (1967). 1 a. AIHARA,K., YAJIMA,G., MORI, H. and HmATA, K., Proc, Fifth Eur. Reg. Conf. Electron Mierose. Manehester 1972, p. 222 (1972). 2. DOUaH~RTY, W. J., Stain Teehnol. 42, 104 (1967). 3. FELDMAN,E. G., J. Cell Biol. 15, 592 (1962). 4. GIBBONS, I. R. and BRADVIELD,J. R. G., Proe. Eur. Reg. Conf. Electron Microse. Stockholm 1956, p. 121 (1957). 5. GORYCKI, M. A., Stain Technol. 41, 169 (1966). 6. HAYAT, M. A., Principles and Techniques of Electron Microscopy, Vol. I, p. 304. Van Nostrand-Reinhold, Princeton, New Jersey, 1970. 7, HuxLEy, H. E. and ZUBAY, G., J. Biophys. Bioehem. Cytol. 11, 273 (1961). 8. KAY, O. H., Techniques for Electron Microscopy, 2nd ed., p. 259. Blackwell, Oxford, 1965. 9. NORMANN, T. C., Stain Teehnol. 39, 50 (1964). 10. NORRIS, G., BANFIELD,W. and CHAHVOUX, H., Sei. Tools 14, 13 (1967). 11. PARSONS,D. F. and DARD~N, E. B., JR., J. Biophys. Biochem. Cytol. 8, 834 (1960). 12. PEACHEY, L. D., J. Biophys. Biochem. Cytol. 5, 511 (1959). 13. ROWD~N, G., J. Mierose. 89, 229 (1969). 14. SJ6STRAND,F. S., Electron Microscopy of Cells and Tissues, Vol. I, p. 297. Academic Press, New York, 1967. 15. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958).
180
J. ULTRASTRUCTURE RESEARCH
42, 180-185 (1973)
An Efficient Standardized Method of Staining Thin Sections for Electron Microscopy M. SJOSTROM,L.-E. THORNELL,and S. HELLSTROM
Department of Anatomy, University of Umed, S-901 87 Umed, Sweden Received June 20, 1972 A simple, rapid, and reproducible method is described for staining of thin sections for electron microscopy. A modified specimen grid box (LKB) is used which holds 100 grids. The sliding cover as well as the bottoms of the compartments of the grid box are bored through. The box is placed in a trough containing the staining solution and then rinsed in beakers with water. The simple and still most widely applied method of staining thin sections for electron microscopy, as described in handbooks on techniques for electron microscopy (6, 8, 14), was introduced by Gibbons and Bradfield in 1956 (4) and was later presented in greater detail by Watson in 1958 (15). The thin sections are collected on a grid, coated with a supporting film or uncoated. Thereafter the grid is either floated on the surface of the staining solution with the section side down or it is immersed in the staining solution. After staining for the necessary time, the sections are immediately rinsed by holding the grid with a forceps and gently flushing the surface of the sections with distilled water. The grid is then placed to dry. This method of staining thin sections has, however, certain disadvantages. The staining solution is to a large extent in contact with air which frequently leads to formation of an insoluble stain precipitate; this in turn may contaminate the sections, e.g., when removing the grids from the stain droplets prior to rinsing process or if some of the staining solution is trapped on the grid or in the forceps when the grid is placed to dry. If many grids are to be stained, the process is time-consuming, especially if multiple sequential staining is performed. Also, reproducibility is rendered more difficult when the staining time, especially if short, is not precisely the same for all grids. Furthermore, the supporting film may be damaged by the repeated use of forceps. This technical communication describes a method of staining up to 100 grids simultaneously. The method also eliminates many sources of artifacts due to precipitated stain, disrupted supporting films, and damaged sections or to varying staining and rinsing times.
STANDARDIZEDSTAINING OF THIN SECTIONS
181
MATERIALS The method is based on the use of a modified specimen grid box, LKB-produkter AB, S-161 25 Bromma, Sweden, No. 4828B, which holds 100 grids in 20 rows of 5 diamondshaped holes (Fig. 1). The sliding cover as well as the bottoms of the plastic box are bored through (Fig. 2). The diameter of the bore-holes is equal to the shortest distance between the parallel edges of the diamond-shaped compartments. A rod-shaped handle, about 40 mm in length, is mounted on the cover. A flat trough just large enough to accommodate the grid box and supplied with a lid is also required (Figs. 3 and 4).
STAINING PROCEDURE W a s h the modified specimen grid box and the trough before use. H o l d the grid box by the handle to avoid contamination by fingerprints. 1. Transfer the grids f r o m the ordinary grid box, where they have been stored since the sectioning procedure, to the staining grid box (Fig. 1). 2. Pour the staining solution into the t r o u g h just prior to the c o m m e n c e m e n t of the staining procedure. 3. Place the box in the t r o u g h (Fig. 3). The staining solution rapidly fills the compartments containing the grids. Cover the t r o u g h with the lid as quickly as possible. 4. After a suitable staining time, remove the lid, and transfer the box to a beaker containing fresh distilled water. Agitate carefully for 5-10 seconds (Fig. 4). 5. Pour rinsing fluid over the end of the tilted box to remove any staining solution that m a y be trapped between the cover and the plastic box (Fig. 5). 6. Finally, wash the box in a second beaker of rinsing solution (Fig. 6). The grid container m a y be dried in several ways, e.g., by placing it on end on a filter paper or some other absorbent material. If multiple sequential staining is desired, simply wash the t r o u g h thoroughly or, preferably, use another one, change the fluids in the beakers, and repeat the steps described above.
FIG. 1. A normal specimen grid box (to the right) compared with the modified grid box (to the left) in which both the sliding cover and the bottoms of the compartments of the plastic box have been bored through. A handle is mounted on the cover. The grids are transferred from the compartments of the ordinary grid box to the corresponding compartments of the staining box. FIG. 2. Schematic drawing showing the grid placed in the diamond-shaped compartment. FIG. 3. The staining grid box has been placed in the trough containing the staining solution which now fills the compartments of the box. Note that the lid (see Fig. 4) is missing for the sake of clarity. Normally the lid covers the trough throughout the staining period. FIG. 4. The box is washed by carefully moving it up and down in a beaker filled with distilled water. FIG. 5. Any staining solution which may be lying between the cover and the plastic box is eliminated by pouring water over the end of the box. FIG. 6. Finally the box is washed in a second beaker of water.
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STANDARDIZED STAINING OF THIN SECTIONS
183
184
SJOSTROM, THORNELL,AND HELLSTR(JlV[
DISCUSSION Many attempts have been made to automate and standardize the different steps in the processing of tissues for electron microscopy (1, 10). These have mostly involved technically advanced and expensive apparatus. Several methods of staining sections mounted on grids other than those discussed in the introduction are in use [cf. Hayat (6)]. Only a few authors, however, have tried to improve the techniques of staining, rinsing, and drying. Gorycki (5) introduced a device which makes it possible to stain 10 grids simultaneously. Feldman (3) presented an apparatus consisting of a series of wells filled with staining solution, at the bottoms of which the grids were placed. However, neither these nor other devices (2, 7, 9, 11-13) seem to be widely adopted, probably because they are not commercially available. Our method of staining thin sections is time-saving and simple to apply. It standardizes the staining; all sections are stained and rinsed for exactly the same time period. This is of advantage when studying serial sections and when comparing the density of structures. The protection provided by the grid box reduces the risk of mechanically ruining sections and supporting films. Any number of successive stains and rinses can be applied without removing the grids from the box. Furthermore, only small amounts (a few milliliters) of stain are required, and the chances of intermingling the grids are minimized. The surface tension of the solutions maintains complete filling of the compartments of the grid box when the box is lifted out of the solutions. The grids are therefore surrounded by staining solution until the moment when the box is immersed in the water rinse and therefore they do not encounter a liquid-air interface at which contaminants may accumulate [see discussion by, e.g., Dougherty (2) and Rowden (13)]. No deposits of precipitated stain are found on the sections, which is perhaps also due to the small area of contact between air and stain. Naturally, the risk of contamination of the sections, by sedimentation of precipitate formed at the surface, is not entirely eliminated. There are, however, other ways to deal with this problem, should it arise. Normann (9) recommended placing grids in envelopes made of filter paper. Similarly, a piece of filter paper may be placed on the cover in contact with the staining solution. The filter paper will then retain any precipitate that may form. If staining solutions which are extremely sensitive to air influence are employed, it is possible to place the grid box and the trough in a suitable chamber gassed with, e.g., nitrogen. The technique of staining thin sections described in this paper has been tested, compared with the standard method, and applied at our laboratory for about one year. The results are fully comparable to carefully performed routine staining of single grids. Naturally, great care must still be exercised when preparing the staining solutions and cleaning glassware and other accessories.
STANDARDIZED STAININGOF THIN SECTIONS
185
This project was supported by grants from the Medical Faculty of Ume~t and from C.-B. Nathhorsts vetenskapliga stiftelse. REFERENCES 1. AIHARA,K., N o ~ z o , K., NAGATA, K., NISm~ZAWA, H. and Suzu~a, K., J. Electronmicrosc. 16, 285 (1967). 1 a. AIHARA,K., YAJIMA,G., MORI, H. and HmATA, K., Proc, Fifth Eur. Reg. Conf. Electron Mierose. Manehester 1972, p. 222 (1972). 2. DOUaH~RTY, W. J., Stain Teehnol. 42, 104 (1967). 3. FELDMAN,E. G., J. Cell Biol. 15, 592 (1962). 4. GIBBONS, I. R. and BRADVIELD,J. R. G., Proe. Eur. Reg. Conf. Electron Microse. Stockholm 1956, p. 121 (1957). 5. GORYCKI, M. A., Stain Technol. 41, 169 (1966). 6. HAYAT, M. A., Principles and Techniques of Electron Microscopy, Vol. I, p. 304. Van Nostrand-Reinhold, Princeton, New Jersey, 1970. 7, HuxLEy, H. E. and ZUBAY, G., J. Biophys. Bioehem. Cytol. 11, 273 (1961). 8. KAY, O. H., Techniques for Electron Microscopy, 2nd ed., p. 259. Blackwell, Oxford, 1965. 9. NORMANN, T. C., Stain Teehnol. 39, 50 (1964). 10. NORRIS, G., BANFIELD,W. and CHAHVOUX, H., Sei. Tools 14, 13 (1967). 11. PARSONS,D. F. and DARD~N, E. B., JR., J. Biophys. Biochem. Cytol. 8, 834 (1960). 12. PEACHEY, L. D., J. Biophys. Biochem. Cytol. 5, 511 (1959). 13. ROWD~N, G., J. Mierose. 89, 229 (1969). 14. SJ6STRAND,F. S., Electron Microscopy of Cells and Tissues, Vol. I, p. 297. Academic Press, New York, 1967. 15. WATSON, M. L., J. Biophys. Biochem. Cytol. 4, 475 (1958).