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Chapter 24 Immunofluorescence Microscopy of Cilia and Flagella
CHAPTER 24
Immunofluorescence Microscopy of Cilia and Flagella M. A. Sanders* and J. L. Salisbury+ 'Imaging Center Genetics and Cell Biology College of Biological Sciences St. Paul, Minnesota 55108 tLaboratory for Cell Biology Department of Biochemistry and Molecular Biology Mayo Clinic Foundation Rochester, Minnesota 55905
1. Introduction 11. General Considerations 111. Reagents and Solutions IV. Immunofluorescence of Ciliated and Flagellated Cells A. Immobilization of Cells B. Fixation C . Permeabilization D. Reduction of Free Aldehydes E. Blocking F. Labeling G. Mounting H. Observation V. Troubleshooting and Variations References
I. Introduction There are a number of excellent articles on fluorescence microscopy and immunofluorescence methods (Asai, 1993; Beltz and Burd, 1989; Giloh and Sedat, 1982; Matsumoto, 1993; Wang and Taylor, 1989). Our aim in this chapter METHODS IN CELL BIOLOGY, VOL. 47 Copyright 0 1995 by Academic Press. Inc. AU rights of reproduction in any form reserved.
163
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is to review the application of methods for immunofluorescence staining of motile ciliated and flagellated cells. We present several fixations and sample preparation protocols that we routinely employ when we are using a previously untested antibody or when we are studying a particular cell type for the first time. Also, we present a general strategy for the simultaneous localization of two separate antigens, as well as nuclear DNA staining in the same cells. Although at least one of these methods generally yields successful results, modifications of the procedures are frequently found though empirical trial and error to result in improved localization.
11. General Considerations The obvious goal of any immunolocalization study is to observe and record the distribution and organization of a particular cellular component in a manner that most closely reflects its distribution in uiuo. Results should be interpreted cautiously, as preparation of the specimen typically involves harsh manipulations of varying degrees of severity. Chemical fixation, with aldehydes or treatment with cold methanol and/or acetone, is commonly employed to immobilize the antigen of interest. Because antibody probes are large molecules, preparative steps typically involve cell permeabilization to allow access to structures of interest. This step may also extract components that otherwise could contribute to unwanted levels of background labeling, possibly reducing “soluble” pools of the antigen itself. To aid in subsequent manipulations,cells are typically immobilized onto a coverglass. This process may affect cell shape, and can result in a flatter specimen that is more amenable to microscopy; however, changes in cell shape can also alter the organization of the protein of interest. Autofluorescence of the specimen, nonspecific labeling by immune serum and secondary antibodies, and specimen thickness also contribute potential problems. Finally, the selective bias of the microscopist to photograph aesthetic or preconceived results can contribute to interpretation prejudice.
111. Reagents and Solutions Highest-purity reagents and deionized water should be used throughout. Adjust the pH to the indicated value at room temperature, and filter all solutions (except methanol) through a 0.2-pm filter (Millipore) prior to use. Microtubule stabilizing buffer (MTSB): Combine 3 mM ethylene glycol bis(Paminoethyl ether)-N,N’-tetraacetic acid (EGTA), 1 mM MgSO,, 25 mM KCl, and 50 mM Na-1,4 piperazinediethanesulfonicacid (Pipes), pH 7.2. MTSB-Triton: Combine MTSB and 0.05% Triton X-100. Formaldehyde fixative: Formaldehyde (30% stock solution) is made fresh by mixing 3 g paraformaldehyde with -8 ml of deionized water and warming the
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solution to 70°C in a water bath located in an exhausted hood. Then add 1 ml of 5 M NaOH dropwise until the solution clears. Cool to room temperature and adjust the final volume to 10 ml. Add 3 ml of the formaldehyde stock to 27 ml of MTSB or MTSB-Triton. If localization of cytoplasmic microtubules is desired we use the MTSB-Triton formulation. Formaldehyde/glutaraldehydefixative: Add glutaraldehyde (8% stock solution, EM grade, Electron Microscopy Sciences, Fort Washington, PA) to the above fixative to a final concentration of 0.1%. MTSB-NH,Cl reducing buffer: Combine MTSB and 50 mM NH,Cl. Phosphate buffer (PB, 10 mM): Add 28 ml KH,PO, and 72 ml K,HPO, to 3900 ml H,O; check pH 7.2. Blocking buffer: Combine 10 mM phosphate buffer, pH 7.2,5% normal goat serum (Gibco BRL, Gaithersberg, MD), 5% glycerol, 1% cold water fish gelatin (Sigma), and 0.04% Na-azide. Aliquot and store at -20°C. Polyethylenimine: Dilute 0.1% polyethylenimine (Sigma) in water. Secondary antibodies: Commercially available secondary antibodies conjugated to rhodamine or fluorescein can be obtained from a number of sources. We routinely use Cappel products (Organon Tecknika, Durham, NC) goat antimouse fluorescein isothiocyanate (F1TC)-conjugated IgG (Catalog No. 55493) or rhodamine-conjugated IgG (Catalog No. 55527) as the secondary for mouse monoclonal primary antibodies, and goat anti-rabbit FITC-conjugated IgG (Catalog No. 55494) or rhodamine-conjugated IgG (Catalog No. 55666) as the secondary for rabbit serum primary antibodies. We find consistent results with secondary antibodies diluted 1 :400 in blocking buffer; however, working dilution should be determined empirically with each new lot or vendor.
IV. Immunofluorescence of Ciliated and Flagellated Cells A. Immobilization of Cells We typically immobilize living or fixed cells onto 12-mm No. 1 round coverslips (Fisher Scientific, Catalog No. 12-545-80) or eight-well epoxy-coated microslides (Carlson Scientific, Peotone, IL) for ease of handling in subsequent steps. The coverslips must first be scrupulously cleaned in 1% 7X detergent, thoroughly rinsed with deionized water, treated with 5 mM ethylenediaminetetraacetic acid (EDTA) for 5-10 minutes, and finally rinsed in deionized water and air-dried. Multiple cover slips can be processed using a coverslip rack (Thomas Scientific, Catalog No. 8542-E30). Place one drop of 0.1% polyethylenimine onto the center of each cleaned cover slip; after 30 seconds, rinse with deionized water and air-dry . Use the polyethylenimine-treated coverslips within 1 hour. Place a drop of cells (washed in fresh culture medium or, for sperm, in phosphate-buffered Tyrode’s salts, Sigma) and allow the cells to settle for 10 minutes. Blot excess suspension and continue immediately to one of the
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fixation protocols listed below. Care must be taken to note the appropriate side of the coverslip for further processing. B. Fixation Two fixation procedures are listed below. The first, -20°C methanol, works well for a majority of the cell types and antibodies that we have tried, and, therefore, it is the general method of choice. Formaldehyde or the combined formaldehyde/glutaraldehyde fixatives work well for particular cell types and epitope/antibody complexes.
1. Cold Methanol Fixation This method simultaneously fixes and permeabilizes the sample. Precool absolute methanol contained in a Coplin jar to -20°C in a freezer. Fix and permeabilize the specimen by submersing the coverslips in the -20°C methanol for 10 minutes. Remove the coverslip, blot excess methanol, air-dry, and rehydrate in phosphate-buffered saline (PBS), three changes, 5 minutes each. Continue procedure at the antibody labeling step (Section IV,F,l).
2. Aldehyde Fixation Prewarm the fixative solution to culture temperature. Submerse the coverslip in the appropriate aldehyde fixative and allow the sample to fix for 30 minutes. This fixation step may contain Triton X-100for simultaneous permeabilization of the cells. C. Permeabilization If the sample has not previously been permeabilized, do so now by submersing the coverslip in MTSB-Triton three times for 5 minutes each.
D. Reduction of Free Aldehydes If the formaldehyde/glutaraldehyde fixative was used, reduce free aldehyde groups by submersing the coverslips in MTSB-NH4C1 reducing buffer, three changes, 5 minutes each, followed by MTSB, three changes, 5 minutes each. Transfer the coverslips through an increasing series of PB :MTSB (33%, 66%, 100% PB). E. Blocking
Block specimens by incubating in blocking buffer for 30 minutes at room temperature.
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F. Labeling 1. Antibody Labeling Dilute the primary antibody of choice in blocking buffer and filter (0.2 pm). Typically, the appropriate primary antibody concentration must be determined empirically. The range for dilution of high-titer sera and monoclonal antibodies is often about 1 : 500 to 1 : 10,000 or greater. Higher serum concentrations may result in unwanted background levels of fluorescence. It is desirable to use preimmune serum produced from the same animal that produced the immune serum, if available, for use as a control treatment to assess the level of nonspecific background labeling. Specific antibodies, affinity purified against the antigen of interest, are also useful for reducing background staining seen in certain immune sera or for particular samples. Incubate the sample in diluted primary antibody for 1 to 4 hours at 37"C, or overnight at 4°C. To avoid dehydration, we typically invert the coverslip over a small drop (-20 pl) of diluted antibody placed on a small piece of Parafilm, and contained in a Petri dish with a moistened filter paper on the inside lid. Following the incubation, carefully remove the coverslip, blot excess antibody solution, and immediately wash in three changes of PBS, 5 minutes each.
2. Secondary Antibody Incubate, as above, in the appropriate secondary antibody diluted 1 : 400 in blocking buffer, for 1-2 hours at 37°C. Carefully remove the coverslip, blot excess antibody solution, and immediately wash in three changes of PBS, 5 minutes each.
3. Double Labeling For double labeling, in which two different antigens are to be localized in the same cells, repeat steps 1 and 2 using the second primary and appropriate secondary antibody preparations. It is necessary to use primary antibodies raised in different species (i.e., rabbit and mouse) and the appropriate secondary antibodies conjugated to distinct fluorochromes to achieve the desired discrimination between the two primary targets. G . Mounting
The mounting medium used must be nonfluorescent, should resist evaporation, and should be formulated to reduce fading of the fluorochrome. A simple antifade mounting medium consisting of 2% N-propyl gallate (Sigma), 30% 0.1 M Tris buffer, pH 9, 70% glycerin (Giloh and Sedat, 1982), and 0.1 pg/ml 4',6-diamidino-2-phenylindole hydrochloride (DAPI, Sigma) to stain DNA has proven effective and easy to use and store (aliquot and freeze at -20°C).
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Following the final PBS wash in step F, 1, briefly rinse the coverslip in deionized water to remove excess salts. Place the inverted coverslip over a small drop of mounting medium on a clean microscope slide, seal the coverslip with nail polish, and allow the nail polish to dry thoroughly before placing the slide on the microscope stage. H. Observation
An epifluorescent microscope equipped with the appropriate excitation and barrier filters for each of the fluorochromes used is required. The best result, in terms of image brightness, is generally obtained with the lowest magnification and highest-numerical-aperture oil immersion objective (i.e., 60X, 1.4 NA) necessary to image the structure of interest. For maximum detail and contrast we generally record black and white images on Hypertech film (Microfluor, Stony Brook, NY), exposed at ASA 800 for fluorescein and 3200 for rhodamine and developed in D-19 developer for 6 minutes at 20°C (Fig. 1, see color plate). For color we record images using a CCD video camera system (VI-470, Optronics Engineering, Goleta, CA) and Image I processing hardware and soft ware (Universal Imaging, West Chester, PA).
V. Troubleshooting and Variations We have found that optimum fixation and success in labeling vary widely for different cell types and antibody probes. The buffer used to wash living cells before fixation and the fixative buffer itself have a dramatic effect on the final quality of labeling. For example, Chlamydomonas cells label best after fixation in Hepes buffer, pH 6.8; vertebrate tissue culture cells label best after fixation in Pipes buffer, pH 7.2; and sperm label best after fixation in phosphate buffer, pH 7.2. Also, divalent cations, particularly calcium, have a dramatic effect on the quality of label, in many cases affecting the stability of the structure under investigation. Although, one could try to determine a priori what the optimal divalent cation composition should be, we have just as often been surprised. The reasons for these differences in fixation and quality of label are not obvious. Therefore, the rule of thumb that we generally apply is to try several buffers, adjusting the pH to as near the culture pH as possible, and to fix samples in the presence of either millimolar Ca2+or EGTA. While aldehyde fixation typically results in the best structural preservation, it may also dramatically affect access and the ability of the antibody to bind to the antigen under investigation. References Asai, D. (1993). Antibodies in cell biology. I n “Methods in Cell Biology” p. 452. Academic Press, San Diego.
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Beltz, B., and Burd, G. (1989). “lmmunocytochemical Techniques, Principles and Practice” p. 181. Blackwell Scientific Publications, Cambridge, MA. Giloh, H . , and Sedat, J. (1982). Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252-1255. Matsurnoto, B. (1993). Cell biological applications of confocal microscopy. I n “Methods in Cell Biology” p. 380. Academic Press, San Diego. Wang, Y.-L., and Taylor, D. (1989). Fluorescence microscopy of living cells in culture. Part A. Fluorescent analogs, labeling cells, and basic microscopy. I n “Methods in Cell Biology” p. 333. Academic Press, San Diego.
Immunofluorescence Microscopy of Cilia and Flagella M. A. Sanders* and J. L. Salisbury+ 'Imaging Center Genetics and Cell Biology College of Biological Sciences St. Paul, Minnesota 55108 tLaboratory for Cell Biology Department of Biochemistry and Molecular Biology Mayo Clinic Foundation Rochester, Minnesota 55905
1. Introduction 11. General Considerations 111. Reagents and Solutions IV. Immunofluorescence of Ciliated and Flagellated Cells A. Immobilization of Cells B. Fixation C . Permeabilization D. Reduction of Free Aldehydes E. Blocking F. Labeling G. Mounting H. Observation V. Troubleshooting and Variations References
I. Introduction There are a number of excellent articles on fluorescence microscopy and immunofluorescence methods (Asai, 1993; Beltz and Burd, 1989; Giloh and Sedat, 1982; Matsumoto, 1993; Wang and Taylor, 1989). Our aim in this chapter METHODS IN CELL BIOLOGY, VOL. 47 Copyright 0 1995 by Academic Press. Inc. AU rights of reproduction in any form reserved.
163
164
M. A. Sanders and J. L. Salisbury
is to review the application of methods for immunofluorescence staining of motile ciliated and flagellated cells. We present several fixations and sample preparation protocols that we routinely employ when we are using a previously untested antibody or when we are studying a particular cell type for the first time. Also, we present a general strategy for the simultaneous localization of two separate antigens, as well as nuclear DNA staining in the same cells. Although at least one of these methods generally yields successful results, modifications of the procedures are frequently found though empirical trial and error to result in improved localization.
11. General Considerations The obvious goal of any immunolocalization study is to observe and record the distribution and organization of a particular cellular component in a manner that most closely reflects its distribution in uiuo. Results should be interpreted cautiously, as preparation of the specimen typically involves harsh manipulations of varying degrees of severity. Chemical fixation, with aldehydes or treatment with cold methanol and/or acetone, is commonly employed to immobilize the antigen of interest. Because antibody probes are large molecules, preparative steps typically involve cell permeabilization to allow access to structures of interest. This step may also extract components that otherwise could contribute to unwanted levels of background labeling, possibly reducing “soluble” pools of the antigen itself. To aid in subsequent manipulations,cells are typically immobilized onto a coverglass. This process may affect cell shape, and can result in a flatter specimen that is more amenable to microscopy; however, changes in cell shape can also alter the organization of the protein of interest. Autofluorescence of the specimen, nonspecific labeling by immune serum and secondary antibodies, and specimen thickness also contribute potential problems. Finally, the selective bias of the microscopist to photograph aesthetic or preconceived results can contribute to interpretation prejudice.
111. Reagents and Solutions Highest-purity reagents and deionized water should be used throughout. Adjust the pH to the indicated value at room temperature, and filter all solutions (except methanol) through a 0.2-pm filter (Millipore) prior to use. Microtubule stabilizing buffer (MTSB): Combine 3 mM ethylene glycol bis(Paminoethyl ether)-N,N’-tetraacetic acid (EGTA), 1 mM MgSO,, 25 mM KCl, and 50 mM Na-1,4 piperazinediethanesulfonicacid (Pipes), pH 7.2. MTSB-Triton: Combine MTSB and 0.05% Triton X-100. Formaldehyde fixative: Formaldehyde (30% stock solution) is made fresh by mixing 3 g paraformaldehyde with -8 ml of deionized water and warming the
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solution to 70°C in a water bath located in an exhausted hood. Then add 1 ml of 5 M NaOH dropwise until the solution clears. Cool to room temperature and adjust the final volume to 10 ml. Add 3 ml of the formaldehyde stock to 27 ml of MTSB or MTSB-Triton. If localization of cytoplasmic microtubules is desired we use the MTSB-Triton formulation. Formaldehyde/glutaraldehydefixative: Add glutaraldehyde (8% stock solution, EM grade, Electron Microscopy Sciences, Fort Washington, PA) to the above fixative to a final concentration of 0.1%. MTSB-NH,Cl reducing buffer: Combine MTSB and 50 mM NH,Cl. Phosphate buffer (PB, 10 mM): Add 28 ml KH,PO, and 72 ml K,HPO, to 3900 ml H,O; check pH 7.2. Blocking buffer: Combine 10 mM phosphate buffer, pH 7.2,5% normal goat serum (Gibco BRL, Gaithersberg, MD), 5% glycerol, 1% cold water fish gelatin (Sigma), and 0.04% Na-azide. Aliquot and store at -20°C. Polyethylenimine: Dilute 0.1% polyethylenimine (Sigma) in water. Secondary antibodies: Commercially available secondary antibodies conjugated to rhodamine or fluorescein can be obtained from a number of sources. We routinely use Cappel products (Organon Tecknika, Durham, NC) goat antimouse fluorescein isothiocyanate (F1TC)-conjugated IgG (Catalog No. 55493) or rhodamine-conjugated IgG (Catalog No. 55527) as the secondary for mouse monoclonal primary antibodies, and goat anti-rabbit FITC-conjugated IgG (Catalog No. 55494) or rhodamine-conjugated IgG (Catalog No. 55666) as the secondary for rabbit serum primary antibodies. We find consistent results with secondary antibodies diluted 1 :400 in blocking buffer; however, working dilution should be determined empirically with each new lot or vendor.
IV. Immunofluorescence of Ciliated and Flagellated Cells A. Immobilization of Cells We typically immobilize living or fixed cells onto 12-mm No. 1 round coverslips (Fisher Scientific, Catalog No. 12-545-80) or eight-well epoxy-coated microslides (Carlson Scientific, Peotone, IL) for ease of handling in subsequent steps. The coverslips must first be scrupulously cleaned in 1% 7X detergent, thoroughly rinsed with deionized water, treated with 5 mM ethylenediaminetetraacetic acid (EDTA) for 5-10 minutes, and finally rinsed in deionized water and air-dried. Multiple cover slips can be processed using a coverslip rack (Thomas Scientific, Catalog No. 8542-E30). Place one drop of 0.1% polyethylenimine onto the center of each cleaned cover slip; after 30 seconds, rinse with deionized water and air-dry . Use the polyethylenimine-treated coverslips within 1 hour. Place a drop of cells (washed in fresh culture medium or, for sperm, in phosphate-buffered Tyrode’s salts, Sigma) and allow the cells to settle for 10 minutes. Blot excess suspension and continue immediately to one of the
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fixation protocols listed below. Care must be taken to note the appropriate side of the coverslip for further processing. B. Fixation Two fixation procedures are listed below. The first, -20°C methanol, works well for a majority of the cell types and antibodies that we have tried, and, therefore, it is the general method of choice. Formaldehyde or the combined formaldehyde/glutaraldehyde fixatives work well for particular cell types and epitope/antibody complexes.
1. Cold Methanol Fixation This method simultaneously fixes and permeabilizes the sample. Precool absolute methanol contained in a Coplin jar to -20°C in a freezer. Fix and permeabilize the specimen by submersing the coverslips in the -20°C methanol for 10 minutes. Remove the coverslip, blot excess methanol, air-dry, and rehydrate in phosphate-buffered saline (PBS), three changes, 5 minutes each. Continue procedure at the antibody labeling step (Section IV,F,l).
2. Aldehyde Fixation Prewarm the fixative solution to culture temperature. Submerse the coverslip in the appropriate aldehyde fixative and allow the sample to fix for 30 minutes. This fixation step may contain Triton X-100for simultaneous permeabilization of the cells. C. Permeabilization If the sample has not previously been permeabilized, do so now by submersing the coverslip in MTSB-Triton three times for 5 minutes each.
D. Reduction of Free Aldehydes If the formaldehyde/glutaraldehyde fixative was used, reduce free aldehyde groups by submersing the coverslips in MTSB-NH4C1 reducing buffer, three changes, 5 minutes each, followed by MTSB, three changes, 5 minutes each. Transfer the coverslips through an increasing series of PB :MTSB (33%, 66%, 100% PB). E. Blocking
Block specimens by incubating in blocking buffer for 30 minutes at room temperature.
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F. Labeling 1. Antibody Labeling Dilute the primary antibody of choice in blocking buffer and filter (0.2 pm). Typically, the appropriate primary antibody concentration must be determined empirically. The range for dilution of high-titer sera and monoclonal antibodies is often about 1 : 500 to 1 : 10,000 or greater. Higher serum concentrations may result in unwanted background levels of fluorescence. It is desirable to use preimmune serum produced from the same animal that produced the immune serum, if available, for use as a control treatment to assess the level of nonspecific background labeling. Specific antibodies, affinity purified against the antigen of interest, are also useful for reducing background staining seen in certain immune sera or for particular samples. Incubate the sample in diluted primary antibody for 1 to 4 hours at 37"C, or overnight at 4°C. To avoid dehydration, we typically invert the coverslip over a small drop (-20 pl) of diluted antibody placed on a small piece of Parafilm, and contained in a Petri dish with a moistened filter paper on the inside lid. Following the incubation, carefully remove the coverslip, blot excess antibody solution, and immediately wash in three changes of PBS, 5 minutes each.
2. Secondary Antibody Incubate, as above, in the appropriate secondary antibody diluted 1 : 400 in blocking buffer, for 1-2 hours at 37°C. Carefully remove the coverslip, blot excess antibody solution, and immediately wash in three changes of PBS, 5 minutes each.
3. Double Labeling For double labeling, in which two different antigens are to be localized in the same cells, repeat steps 1 and 2 using the second primary and appropriate secondary antibody preparations. It is necessary to use primary antibodies raised in different species (i.e., rabbit and mouse) and the appropriate secondary antibodies conjugated to distinct fluorochromes to achieve the desired discrimination between the two primary targets. G . Mounting
The mounting medium used must be nonfluorescent, should resist evaporation, and should be formulated to reduce fading of the fluorochrome. A simple antifade mounting medium consisting of 2% N-propyl gallate (Sigma), 30% 0.1 M Tris buffer, pH 9, 70% glycerin (Giloh and Sedat, 1982), and 0.1 pg/ml 4',6-diamidino-2-phenylindole hydrochloride (DAPI, Sigma) to stain DNA has proven effective and easy to use and store (aliquot and freeze at -20°C).
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Following the final PBS wash in step F, 1, briefly rinse the coverslip in deionized water to remove excess salts. Place the inverted coverslip over a small drop of mounting medium on a clean microscope slide, seal the coverslip with nail polish, and allow the nail polish to dry thoroughly before placing the slide on the microscope stage. H. Observation
An epifluorescent microscope equipped with the appropriate excitation and barrier filters for each of the fluorochromes used is required. The best result, in terms of image brightness, is generally obtained with the lowest magnification and highest-numerical-aperture oil immersion objective (i.e., 60X, 1.4 NA) necessary to image the structure of interest. For maximum detail and contrast we generally record black and white images on Hypertech film (Microfluor, Stony Brook, NY), exposed at ASA 800 for fluorescein and 3200 for rhodamine and developed in D-19 developer for 6 minutes at 20°C (Fig. 1, see color plate). For color we record images using a CCD video camera system (VI-470, Optronics Engineering, Goleta, CA) and Image I processing hardware and soft ware (Universal Imaging, West Chester, PA).
V. Troubleshooting and Variations We have found that optimum fixation and success in labeling vary widely for different cell types and antibody probes. The buffer used to wash living cells before fixation and the fixative buffer itself have a dramatic effect on the final quality of labeling. For example, Chlamydomonas cells label best after fixation in Hepes buffer, pH 6.8; vertebrate tissue culture cells label best after fixation in Pipes buffer, pH 7.2; and sperm label best after fixation in phosphate buffer, pH 7.2. Also, divalent cations, particularly calcium, have a dramatic effect on the quality of label, in many cases affecting the stability of the structure under investigation. Although, one could try to determine a priori what the optimal divalent cation composition should be, we have just as often been surprised. The reasons for these differences in fixation and quality of label are not obvious. Therefore, the rule of thumb that we generally apply is to try several buffers, adjusting the pH to as near the culture pH as possible, and to fix samples in the presence of either millimolar Ca2+or EGTA. While aldehyde fixation typically results in the best structural preservation, it may also dramatically affect access and the ability of the antibody to bind to the antigen under investigation. References Asai, D. (1993). Antibodies in cell biology. I n “Methods in Cell Biology” p. 452. Academic Press, San Diego.
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Beltz, B., and Burd, G. (1989). “lmmunocytochemical Techniques, Principles and Practice” p. 181. Blackwell Scientific Publications, Cambridge, MA. Giloh, H . , and Sedat, J. (1982). Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science 217, 1252-1255. Matsurnoto, B. (1993). Cell biological applications of confocal microscopy. I n “Methods in Cell Biology” p. 380. Academic Press, San Diego. Wang, Y.-L., and Taylor, D. (1989). Fluorescence microscopy of living cells in culture. Part A. Fluorescent analogs, labeling cells, and basic microscopy. I n “Methods in Cell Biology” p. 333. Academic Press, San Diego.