免疫组化

*Edited by Oliver Hobert. WormMethods editor, Victor Ambros. Last revised February 16, 2005. Published June 19, 2006. This chapter should be cited as: Duerr, J. S. Immunohistochemistry (June 19, 2006), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.105.1, http://www.wormbook.org. Copyright: © 2006 Janet S. Duerr. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. §To whom correspondence should be addressed. E-mail: duerr@ohio.edu Immunohistochemistry* Janet S. Duerr§, Department of Biological Sciences, Ohio University, Athens, OH 45701 USA Table of Contents 1. Introduction ............................................................................................................................ 1 1.1. Immunocytochemistry .................................................................................................... 2 1.2. Western blot analysis ..................................................................................................... 2 1.3. General comments ......................................................................................................... 3 2. Protocols and procedures ........................................................................................................... 6 2.1. Protocol 1: Antigen preparation ........................................................................................ 6 2.2. Protocol 2: Peptide coupling ............................................................................................ 7 2.3. Protocol 3: Chicken antibody purification .......................................................................... 8 2.4. Protocol 4: Affinity purification ..................................................................................... 10 2.5. Protocol 5: Fixation conditions ...................................................................................... 14 2.6. Protocol 6: Freeze-crack ............................................................................................... 16 2.7. Protocol 7: Tube fixation .............................................................................................. 24 2.8. Protocol 8: Bouin's tube fixation .................................................................................... 27 2.9. Protocol 9: Peroxide tube fixation ................................................................................... 29 2.10. Protocol 10: Picric acid + glutaraldehyde fixation ............................................................ 32 2.11. Protocol 11: Depletion of primary antibody .................................................................... 35 2.12. Protocol 12: Cleaning secondary antibody ...................................................................... 37 2.13. Protocol 13: Staining slides ......................................................................................... 39 2.14. Protocol 14: Staining tube-fixed worms ......................................................................... 45 2.15. Protocol 15: Worm protein preparation .......................................................................... 48 2.16. Protocol 16: Worm protein gel ..................................................................................... 51 2.17. Protocol 17: Western transfer ....................................................................................... 55 3. References ............................................................................................................................ 60 1. Introduction Immunohistochemistry in C. elegans includes two major classes of techniques: immunocytochemical localization of proteins in situ and Western blot analysis of purified proteins. In this section, we will discuss a variety of techniques for immunocytochemistry, as well as simple Western blotting techniques for worm proteins. 1 Immunocytochemistry provides the most direct method for identifying both the cellular and subcellular distribution of your protein. Although GFP or other translational or transcriptional constructs can provide a relatively rapid indication of gene expression or protein distribution, there are a number of potential problems with these techniques (Mello and Fire, 1995). These include abnormal regulation of expression from multi-copy transgenic arrays and abnormal localization of the protein due to over-expression or the presence of the tag. If possible, the predicted distribution of your protein should be confirmed with the use of antibodies in situ. Specific antibodies also allow you to examine protein modifications and expression in vivo. Although antibodies can be very useful tools, generating a specific antibody protein can be difficult, time-consuming, and expensive (see Harlow and Lane, 1988). Unfortunately, except for antibodies to a few highly conserved proteins (e.g., actin and tubulin), most antibodies generated against specific vertebrate or invertebrate proteins do not cross-react specifically with their C. elegans homologs. Furthermore, when you specifically generate an antibody against a C. elegans protein, the antibody may only work with a subset of immune techniques, e.g., Westerns but not in situ or vice versa. A brief Antigen preparation protocol highlights some considerations when designing antigens. Whenever you use antibodies, it is important to test for specificity. In addition to the standard controls, such as pre-absorbance of your antibody, worm-specific methods may be available to test antibody specificity. If there are null mutants or RNAi worms for the gene and protein of interest, then compare antibody staining (of Westerns or in situ) in those strains with staining in wild-type worms. Nulls can also be used to affinity deplete your serum (see Depletion of primary antibody protocol). On the other hand, if there are transgenic over-expressing lines for the protein of interest, those can be used as positive controls. Generally, you will need to do some sort of affinity depletion before you can detect specific staining of Westerns or worms (see Affinity purification protocol). 1.1. Immunocytochemistry C. elegans provides specific rewards and particular challenges for in situ analysis of protein distribution. If you have an antibody that works in situ, you can examine the distribution of the protein in the entire organism at all stages relatively rapidly. Other than the intestine, most tissues have relatively low levels of autofluorescence, so many different dyes and secondary antibodies may be used, allowing triple labeling of antibodies or more. On the other hand, immunocytochemistry in worms is relatively difficult due to problems of access and size. Both the egg shell of embryos and the cuticles of larvae and adults are relatively impermeable and must be disrupted chemically or mechanically (or both) for the antibody to have access to the internal cells and proteins. The small size of the individual cells and their compact architecture can make visualization of the cellular and subcellular distribution of proteins in some cells quite difficult. A confocal fluorescent microscope or a wide-field fluorescent microscope with de-convolution software can provide increased spatial resolution. When detailed examination of subcellular distribution is necessary, then one can attempt the supreme challenge of immuno-electron microscopy. For a discussion of electron microscopy and immuno-EM in C. elegans (not covered in this section), see the Worm Atlas website. There are several different techniques used to allow antibodies access to the tissue of interest. If you are specifically interested in the distribution of a protein within the cells of the gonad, the very early embryo, or the intestine, then the appropriate tissue can be dissected out of the adult (see Freeze-crack protocol). To examine protein in a larva or adult, the two general methods are to 1) collect worms in small tubes of liquid and to freeze and chemically treat them to disrupt their cuticles (see Tube fixation, Bouin's tube fixation, Peroxide tube fixation, and Picric acid + glutaraldehyde fixation protocols) or 2) compress and freeze worms between slides and mechanically pull off the cuticle layer (see Freeze-crack protocol). Different fixation conditions differentially preserve antigenicity and tissue morphology. The Fixation conditions protocol discusses how to determine the optimum fixation conditions for a particular antibody and protein combination. You can use a control antibody such as anti-DNA or anti-actin to monitor accessibility and morphology with the different fixation conditions. 1.2. Western blot analysis Western blot analysis is useful for several reasons. It can be used to examine the relative levels of expression of different size forms of your protein, such as those due to alternative splicing. It can help identify post-translational modifications such as phosphorylation or glycosylation. Western blot analysis can be used to Immunohistochemistry 2 screen for changes in protein expression under different conditions or in different mutant backgrounds. Changes in protein expression may also be seen with immunocytochemistry (and examined in a cell-specific way). But, generally antibodies do not distinguish between protein variants in situ unless specifically designed to do so. And some antibody works well for Westerns but not in situ. Preparation of denatured protein from C. elegans is relatively straight forward, since simple boiling of adults or embryos in a typical protein buffer frees many proteins from the body (see Worm protein preparation protocol). A major difficulty with Western blot analysis arises from the fact that the entire worm is usually used for protein preparation. Thus, even if your protein is of fair abundance in a particular tissue, e.g., the nervous system, its abundance relative to total worm protein may be quite low. Therefore, sensitive detection methods are generally required (see Western transfer protocol). If you generated your antibody using a fusion protein or a coupled peptide, then first test your antibody for staining of a Western Blot using a very low concentration of this protein. If you can not detect the protein in this purified condition, you are unlikely to be able to detect it in a complex mixture of worm proteins. The antibody may still work on worms in situ, since the antigen will be in a different conformation in the fixed worm than in the protein gel. When you test an antibody on a worm preparation, it is helpful if you can use a Western blot from a tissue or strain that is enriched for the protein. For example, if the protein is enriched in a particular stage, then preparation of synchronous populations is recommended. If a particular enriched tissue can be dissected out fairly rapidly (e.g., the gonad), that is also recommended as the starting preparation. If you have a transgenic strain or mutant that over-expresses your protein, then use it to test for specificity of antibody binding in Westerns. 1.3. General comments C. elegans cell culture (Christensen et al., 2002) provides novel tools for immunohistochemistry. Cell culture provides an easy method to test antibodies for staining of cells without concerns about permeability and accessibility (see Fixation conditions protocol). It also may provide an enriched source of particular cell types (e.g., sorted GFP-positive cells) for use in Western analysis. Despite this, the intact organism continues as the major focus of immunohistochemical techniques, as described in the following protocols. The figures illustrate antibody staining of C. elegans using the protocols covered in this section. The fixation conditions range from a very "light" fix (Figure 1, Protocols 6 and 13), to very "hard" fixes, followed by permeabilization by collagenase treatment (Figure 2, Protocols 7 and 14) or other chemical permeabilization (Figure 3, Protocols 8 and 14). Each antibody has its own optimum fixation conditions, see Protocol 5 for a discussion of how to determine those conditions. For staining in wild type versus transgenic strains, see Figure 4. Immunohistochemistry 3 Figure 1. Light fixation and staining of multiple antigens in the head of an adult OH99 mgIs18 [Pttx-3GFP] prepared using Protocol 6. Freeze-crack. (A) Staining of the vesicular monoamine transporter (VMAT = CAT-1), shown in red. This antigen is found in synaptic vesicles of monoaminergic neurons.(B) DNA, white, was labeled with DAPI. (C) The vesicular acetylcholine transporter (VAChT = UNC-17), found in cholinergic synaptic vesicles, is shown in blue. (D) Staining with an anti-GFP antibody is shown in green. The GFP fills the cytoplasm of the AIY neurons. (E) Merged image of anti-VMAT (red), anti-VAChT (blue), and anti-GFP (green). (F) Merged image of anti-VMAT (red), anti-GFP (green) and DAPI (blue). Fixation was for 2 minutes in methanol followed by 4 minutes in acetone. Staining followed Protocol 13: Staining slides. Primary antibodies were goat anti-VMAT (Goat 258 polyclonal antibody purified with Protocol 4: Affinity purification), rabbit anti-GFP (Molecular Probes) and mouse anti-VAChT (Monoclonal 1403). Secondary antibodies were Cy3-conjugated donkey anti-goat, Oregon Green 488-conjugated donkey anti-rabbit, and Cy5-conjugated donkey anti-mouse. See Protocol 13: Staining slides for a discussion of anti-GFP and secondary antibodies. mgIs18 [Pttx-3::GFP] was a gift of O. Hobert; unc-104(e1265) was provided by the CGC. Images are maximum projections of confocal series, contrast enhanced in Adobe Photoshop®. Anterior to left, ventral down, scale bar is 10 mm. Immunohistochemistry 4 Figure 2. Hard fixation in tubes to visualize neurotransmitters. Serotonin (red) and GFP (green) were labeled in the head of an adult RM2304 unc-104(e1265); mgIs18 [Pttx-3::GFP]. The bright red cells are the NSMs; the bright green cells are the AIYs. Worms were prepared using Protocol 7: Tube fixation with fixation for 24 hours in 4% formaldehyde, followed by incubation in collagenase for 6.5 hours. Staining was done using Protocol 14: Staining tube-fixed worms. Primary antibodies were rabbit anti-serotonin (H. Steinbusch, Free University) and mouse anti-GFP (Molecular Probes monoclonal 3E6); secondary antibodies were Cy3-conjugated donkey anti-rabbit and Oregon Green 488-conjugated donkey anti-mouse. mgIs18 [Pttx-3::GFP] was a gift of O. Hobert; unc-104(e1265) was provided by the CGC. Images are maximum projections of confocal series, contrast enhanced in Adobe Photoshopâ. Anterior to left, ventral down, scale bar is 10 mm. Figure 3. Bouin's tube fixation for muscle preservation. (A) Actin was labeled in a wild-type L2 larva fixed using Protocol 8: Bouin's tube fixation with 1 hour in Bouin's fixative with methanol and b-mercaptoethanol, followed by incubation in Borate Tris buffers. Staining was using Protocol 14: Staining tube-fixed worms with mouse anti-actin (Chemicon monoclonal C4), followed by Oregon Green 488-conjugated donkey anti-mouse. (B) Talin was labeled in a wild-type embryo with rabbit B547 anti-talin (gift of Bob Barstead and Gary Moulder) and Cy3-conjugated donkey anti-rabbit. (C) Double staining of talin, stained with rabbit B547, and vinculin, stained with monoclonal antibody MH24 (gift of Russ Francis) in a wild-type adult. Secondary antibodies were Oregon Green 488 donkey anti-rabbit and Cy3 donkey anti-mouse. Maximum projection of overlapping confocal series, contrast enhanced in Adobe Photoshopâ. All scale bars are 10 mm. Immunohistochemistry 5 Figure 4. Staining in wild-type versus transgenic strains. The transgenic strain RM1810 cha-1(md39); mdEx10 [F57G7] contains multiple copies of the cosmid that contains the cha-1 and unc-17 genes. This strain produces abnormally high levels of the ChAT and UNC-17/VAChT proteins. It is useful for testing the specificity of anti-ChAT and anti-VAChT antibody staining in situ, for increased ease of cell identification, and also for Westerns (see Protocol 17: Western transfer). (A) Staining of the VAChT, shown in red, in RM1810. (B) The same worm stained with DAPI to show nuclei. (C) A higher magnification view of the head of the same individual. (D) VAChT staining in a wild-type N2 shows the typical synaptic localization of the protein. Note that over-expression of VAChT leads to mislocalization of the protein in neuronal somas (compare panels C and D). The RM1810 and wild-type worms were prepared using Protocol 6: Freeze-crack. Fixation was for 2 minutes in methanol followed by 4 minutes in acetone. Staining followed Protocol 13: Staining slides, with mouse anti-VAChT (Monoclonal 1403) as the primary antibody, followed by Cy3-conjugated donkey anti-mouse. DNA was labeled by addition of DAPI in the mounting medium. Maximum projection of overlapping confocal series, contrast enhanced in Adobe Photoshop®. Anterior to left, ventral down, scale bar is 10 mm. 2. Protocols and procedures 2.1. Protocol 1: Antigen preparation 2.1.1. General comments For a thorough discussion of antigens and antibody generation, see Harlow and Lane (1988). 2.1.1.1. Antibody production There are a number of companies that will assist you in your antibody production, from the generation of fusion proteins or peptide antigens through serum purification. See the following for suggestions for peptide design and coupling (see Peptide coupling protocol) and affinity purification (see Antibody purification protocol). 2.1.1.2. Animal host The most frequently immunized animal is the rabbit. If you plan to do any double labeling, try generating your antibodies in one or two different species. Chickens are recommended: they are relatively inexpensive, the collection of antibody in eggs is humane, and they often generate robust immune responses. It is generally more economical to have the company deliver the eggs to you rather than the purified serum. Chicken antibodies are easy and inexpensive to partially purify from the egg yolks (see Chicken antibody purification protocol). 2.1.1.3. Fusion proteins Generation of fusion proteins for immunization is much more laborious than buying peptides, but immunization with fusion proteins is more likely to lead to useful antibodies. If you have a company generate the Immunohistochemistry 6 of your serum. Green Fluorescent Protein can be a wonderful protein to use in translational fusions (see Reporter gene fusions section of WormMethods). If expression of a translational GFP fusion leads to rescue of the mutant phenotype, then the location of GFP fluorescence in the rescued animals is likely to correspond at least partially to the normal localization of your protein. Of course, the localization should be checked with immunocytochemical localization of the endogenous protein in a wild-type animal. If your protein is not functional or stable in a GFP fusion protein, there are a number of very small epitope tags such as 6-His and Myc that can be fused to your protein and used for preliminary localization studies (Roehl et al., 1996). 2.1.1.4. Peptides Immunization with synthetic peptides is the quickest and easiest way to try and generate antibodies, although it is not always productive. Companies will predict immunogenic peptides or you can design the peptides yourself (http://immunax.dfci.harvard.edu/Tools/antigenic.html). If they are potentially immunogenic, then N-terminal or C-terminal peptides are recommended. Be sure to order enough peptide for testing, affinity purification, and affinity depletion of your serum. Use at least two differe