Tips for designing a successful IHC experiment
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Although immunohistochemistry (IHC) is a relatively straightforward experimental method, there are a number of variables that have to be optimized for each IHC study.
Here, we summarize some of the variables that should be considered to ensure consistent and reproducible results.
| Variable | Factors to consider |
| Antigen | Species, expression level, sample type |
| Epitope | If mapped - dependence on post-translational modification |
| Appropriate controls | Positive and negative controls - no primary antibody, isotype control, absorption control, tissue type control |
| Sample preparation | Fixed or frozen |
| Fixation method | Perfusion or immersion (with or without freezing) |
| Fixative | Formaldehyde, alcohols or acetone (including concentration, pH, temperature, incubation time and diluents) |
| Blocking steps | Protein blocking (e.g., with serum or BSA) to prevent non-specific binding, blocking of endogenous enzymes and biotin where required |
| Antigen retrieval | Proteolytic-Induced Epitope Retrieval (PIER) or Heat-Induced Epitope Retrieval (HIER) |
| Detection method | Direct or indirect (with or without amplification) |
| Detection complex | ABC, LSAB, polymer or micro-polymer |
| Primary antibody | Monoclonal or polyclonal |
| Secondary antibody | Species and label |
| Labeling method | Chromogenic or fluorescent |
| Label | Fluorochromes: depending on desired spectral properties Chromogens: 3,3-diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), 3,3’,5,5’-tetramethylbenzidine (TMB), New Fuchsin, 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/ NBT) |
| Counterstain | Fluorescent e.g., DAPI, DRAQ5™, DRAQ7™, Nuclear green, Hoechst Chromogenic: hematoxylin and eosin or histology special stain |
| Mounting reagent | Fluorescent: anti-fade aqueous mounting medium Chromogenic: organic/aqueous mounting medium |
| Visualization and analysis | Fluorescence microscope or standard microscope; analysis by eye or software-based imaging |