IHC guide v3 952x200px

Tips for designing a successful IHC experiment

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. 

VariableFactors to consider
AntigenSpecies, expression level, sample type
EpitopeIf mapped - dependence on post-translational modification
Appropriate controlsPositive and negative controls - no primary antibody, isotype control, absorption control, tissue type control
Sample preparationFixed or frozen
Fixation methodPerfusion or immersion (with or without freezing)
FixativeFormaldehyde, alcohols or acetone (including concentration, pH, temperature, incubation time and diluents)
Blocking stepsProtein blocking (e.g., with serum or BSA) to prevent non-specific binding, blocking of endogenous enzymes and biotin where required
Antigen retrievalProteolytic-Induced Epitope Retrieval (PIER) or Heat-Induced Epitope Retrieval (HIER)
Detection methodDirect or indirect (with or without amplification)
Detection complexABC, LSAB, polymer or micro-polymer
Primary antibodyMonoclonal or polyclonal
Secondary antibodySpecies and label
Labeling methodChromogenic 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 reagentFluorescent: anti-fade aqueous mounting medium
Chromogenic: organic/aqueous mounting medium
Visualization and analysisFluorescence microscope or standard microscope; analysis by eye or software-based imaging

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