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Reagents can be applied manually by pipette or the sequence of the protocol can be adapted to automated and semi-automated systems if these are available.
All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is an adequate chamber, just as long as the slides are kept off the paper and can lay flat so that the reagents don't drain off! A good solution is to cut a plastic serological pipette into lengths to fit your incubation chamber. Glue them in pairs to the bottom of the chamber, with the 2 individual pipette tubes of each pair being placed about 4 cm apart. This provides a level and raised surface for the slides to rest on away from the wet tissue paper.
Dilutions of the primary and secondary antibody are listed on the datasheets or are determined by testing a range.
Adjust dilutions appropriately from the results obtained. Adhere strictly to all incubation times in the protocol.
For enzymatic methods, horseradish peroxidase (HRP) or alkaline phosphatase (AP) are the most commonly used enzymes. There are a number of chromogens used with these enzymes (see note x).
If necessary, perform antigen retrieval before commencing with immunostaining.
For enzymatic detection (HRP or AP secondary conjugates):
For fluorescent detection:
Notes and tips:
i. The use of 0.025% Triton X-100 in the TBS helps to reduce surface tension, allowing reagents to cover the whole tissue section with ease. It is also believed to dissolve Fc receptors, therefore reducing non-specific binding. We recommend TBS rather than PBS to get a cleaner background.
ii. The secondary antibody may cross react with endogenous immunoglobulins in the tissue. This is minimized by pre-treating the tissue with normal serum from the species in which the secondary was raised. The use of normal serum before the application of the primary also eliminates Fc receptor binding of both the primary and secondary antibody. BSA is included to reduce non-specific binding caused by hydrophobic interactions.
iii. The primary antibody should be diluted to the manufacturer’s recommendations or to a previously optimized dilution. Most antibodies will be used in IHC-P at a concentration between 0.5 and 10 μg/ml. Make sure the primary antibody is raised in a species different from the tissue being stained. If, for example, you had mouse tissue and your primary antibody was raised in a mouse, an anti-mouse IgG secondary antibody would bind to all the endogenous IgG in the mouse tissue, leading to high background. Use of mouse monoclonals on mouse tissue is discussed in our mouse-on-mouse protocol.
iv. Overnight incubation allows antibodies of lower titer or affinity to be used by allowing more time for the antibodies to bind. Also, regardless of the antibody's titer or affinity for its target, once the tissue has reached saturation point no more binding can take place. Overnight incubation ensures that this occurs.
v. H2O2 suppresses endogenous peroxidase activity and therefore reduces background staining. To check for the presence of endogenous peroxidases, incubate a tissue slide after re-hydration in a solution of DAB. If areas of the section appear brown under the microscope, a blocking step should help reduce this staining. Some epitopes are modified by peroxide, leading to reduced antibody-antigen binding. Incubating sections with peroxide after the primary incubation avoids this problem. Peroxide can be diluted in TBS or water. Some laboratories use methanol which is useful for blood smears or other peroxidase-rich tissues; peroxide diluted in methanol tends to reduce damage to the tissue caused by the reaction in aqueous solutions. For other tissue, we recommend diluting in TBS or water. Reduced binding of some antibody-antigen pairs, in particular cell surface proteins, has been observed after methanol/peroxide incubation. (If using AP or fluorescent detection, omit peroxidase quenching as it only applies to HRP conjugates).
vi. Develop the colored product of the enzyme with the appropriate chromogen. The choice depends on which enzyme label you are using, the colored end product you prefer and whether you are using aqueous or organic mounting media (see note ix for further details).
vii. Some commonly used counterstains are hematoxylin (blue), nuclear fast red, or methyl green. When using fluorescent detection, DAPI (blue) or propidium iodide/PI (red) can be used to counterstain.
viii. DAB is a suspected carcinogen. Wear the appropriate protective clothing. Use chloros to stop the DAB reaction in a fume hood overnight (it produces noxious fumes when chloros is added) and dispose of it according to laboratory guidelines. If using AP, add 0.24 mg/ml Levamisole (Sigma L9756) to the chomogen solution. Levamisole suppresses endogenous phosphatase activity and therefore reduces background staining.
ix. If using AEC, Fast Red, INT or any other aqueous chromogen then do not forget that they are alcohol soluble. Use a suitable aqueous mounting media. Do not dehydrate and clear!
x. Dehydrate and clear sections developed using DAB, New Fuchsin, Vega Red, NBT, TNBT or any other organic chromogen developed sections by sending them through the rehydration steps listed in our deparaffinization protocol. Mount sections in a suitable organic mounting medium. Sections mounted in organic mounting media have a better refractive index than those mounted in aqueous mounting media. This means that the image seen down the microscope will be sharper and clearer if an organic mounting medium is used.
Materials and reagents
Peroxidase block (40 ml)
20 ml 0.2M Phosphate Buffer
8 ml methanol
80 µl Triton-X100
2 ml Hydrogen Peroxide
Make up to 40 ml with ddH2O
0.1M Phosphate buffer
1% serum from secondary antibody host species
To achieve a stronger signal, various strategies have been developed to add more enzyme or fluorophore to the target of interest.
a) Avidin-biotin complex (ABC)
This technique, developed by Su-Ming Hsu and colleagues (J Histochem CytoChem. 1981 Apr 29 (4): 577-80), utilizes the high affinity of avidin, a protein found in chicken egg white, for biotin, an enzyme co-factor in carboxylation reactions. Avidin has four binding sites for biotin and binding is essentially irreversible. In brief, the primary antibody is bound to the protein of interest. A biotinylated secondary antibody is then bound to the primary antibody. In a separate reaction, a complex of avidin and biotinylated enzyme is formed by mixing the two in a ratio that leaves some of the binding sites on avidin unoccupied. This complex is then incubated with the tissue section after the antibody incubations. The unoccupited biotin-binding sites on the complex bind to the biotinylated secondary antibody. The result is more enzyme attached to the target than is possible using an enzyme-conjugated secondary or primary antibody.
The components of the avidin-biotin complex are commercially available in kits that provides the two reagents and instructions for combinging them in the optimal ratio. The complex can be used with any of our biotinylated antibodies. Once concern is the presence of endogenous biotin in tissues such as kidney, liver, brain, prostate, colon, intestines, and testes, which can bind the avidin-biotin complex leading to background staining (Wand and Pevsner, Cell Tissue Res. 1999 Jun; 296(3): 511-6).
b) Labeled streptavidin biotin (LSAB)
This method is similar to ABC in that it utilizes the interation of streptavidin (similar to avidin in binding affinity) and biotin. The primary antibody is followed by a biotinylated anti-lg secondary antibody, followed by streptavidin conjugated to an enzyme or fluorophore. Streptavidin produces less non-specific background staining than avidin since it is non-glycosylated (unlike avidin), and so shows no interaction with lectins or other carbohydrate-binding proteins. For a comparison to ABS in which LSAB was shown to be 4-8 times more sensitive, see Giorno R, Diagno Immunol. 1984;2(3):161-6.
c) HRP polymer
Both avidin-biotin methods (ABC and LSAB) are losing favor to new polymer-enzyme-antibody products that consist of a secondary antibody (e.g. anti-mouse and/or rabbit IgG) attached to a polymer-enzyme complex. One step is eliminated compared to the AB methods and the issue of endogenous biotin is avoided.
A further improvement on polymer methods, EXPOSE detection kits for IHC use small linker-based detection modules that can penetrate tissues better than the large complexes used in conventional polymer-based detection, resulting in higher sensitivity.
d) Tyramide signal enhancing (TSE)
One of the most effective amplification procedures is the patented and licensed method, TSE (also known as TSA or CSA). It is particularly useful for detection of relatively sparse antigens that other systems have difficulty detecting, and for improving results obtained with poorly-performing antibodies.
The method relies on a peroxidase-catalyzed reaction to covalently attach the tyramide portion of tyramine-protein conjugates to the area around the protein of interest, after first applying a primary antibody and secondary-HRP conjugate. The covalently attached protein cannot be washed off, even if the slides are treated to remove the antibodies, since the tyramide bond is covalent. To obtain a signal, an antibody-enzyme or fluorophore conjugate is directed against the protein portion of the tyramine-protein conjugate.
The disadvantages of the procedure are the expense of the kits and the time required to perform the multiple steps.