Designing a Successful IHC/ICC Experiment

Immunohistochemistry (IHC) and immunocytochemistry (ICC) are techniques employed to localize antigen expression and are dependent on specific epitope-antibody interactions. IHC refers to the use of tissue sections, whereas ICC describes the use of cultured cells or cell suspensions. In both methods, positive staining is visualized using a molecular label, which can be fluorescent or chromogenic. Briefly, samples are fixed to preserve cellular integrity, and then subjected to incubation with blocking reagents to prevent non-specific binding of the antibodies. Samples are subsequently incubated with primary and secondary antibodies, and the signal is visualized for microscopic analysis.

Technically, IHC/ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be recognized and optimized for each individual IHC/ICC study. The most challenging aspect of these techniques is determining the experimental conditions necessary to generate a strong and specific signal for each antigen of interest. For example, detection of an abundant protein in cultured cells may require a short fixation period, minimal blocking, and may be compatible with direct visualization using a fluorochrome-conjugated primary antibody. In contrast, detection of a phosphorylation-dependent epitope in a section of frozen tissue may require antigen retrieval and be dependent on amplified chromogenic visualization. This section of our website provides information on the IHC/ICC variables listed below and is intended to assist in the design, optimization, and troubleshooting of IHC/ICC experiments.

Variables for Experimental Design & Optimization

Variable Factors
Antigen Species, expression levels, sample types, & subcellular location
Epitope Dependence on conformation or post-translation modification
Sample Type Tissue or cells
Sample Preparation Tissue: embedded or frozen
Cells: adhered cell cultures or cell suspensions
Appropriate Controls No primary antibody, isotype control, absorption control, tissue type control
Fixation Method Perfusion or immersion (with or without freezing)
Fixative* Formaldehyde, alcohols, or acetone
Blocking Reagent* Normal serum, bovine serum albumin (BSA), or non-fat milk
Antigen Retrieval* Protease-induced Epitope Retrieval (PIER) or Heat-induced Epitope Retrieval (HIER)
Detection Method Direct or indirect (with or without amplification)
Primary Antibody* Monoclonal or polyclonal
Secondary Antibody* Species and label
Labeling Method Fluorescence or chromogenic
Label Fluorochromes and spectral properties
Chromogens: 3,3' Diaminobenzidine (DAB), 3-amino-9-ethylcarbazole (AEC)
Counterstain* Fluorescence: 4,6'-diamidino-2-phenylindole (DAPI)
Chromogenic: hematoxylin
Mounting Reagent Fluorescence: anti-fade mounting medium
Chromogenic: aqueous mounting medium
Visualization & Analysis Fluorescence microscope or standard light microscope
* These variables require optimization of additional factors including concentration, pH, temperature, incubation time, and diluent.
This table is not intended to be comprehensive but represents the most commonly applied IHC/ICC methods at R&D Systems.



Examples of Fluorescence Detection by IHC & ICC. A. Fluorescence detection by IHC was used to visualize V-set and IG domain-containing protein 1 (VSIG1) in a frozen section of human testis using anti-human VSIG1 affinity-purified polyclonal antibody (Catalog # AF4818). Tissue was stained using NorthernLightsTM-557 anti-sheep IgG secondary antibody (Catalog # NL010; red) and nuclei were counterstained with DAPI (blue). B. Fluorescence detection by ICC was used to visualize Glial Fibrillary Acidic Protein (GFAP) in differentiating cultures of rat cortical stem cells using anti-human GFAP affinity-purified polyclonal antibody (Catalog # AF2594). The cells were stained using anti-sheep NorthernLights-557 secondary antibody (Catalog # NL010; red) and nuclei were counterstained with DAPI (blue).




Examples of Chromogenic Detection by IHC & ICC. A. Chromogenic detection by IHC was used to visualize Interferon Regulator Transcription Factor 8 (IRF8) in a paraffin-embedded section of human lymphoma tissue using anti-human IRF8 affinity-purified polyclonal antibody (Catalog # AF5117). Tissue was stained using R&D Systems anti-sheep HRP-DAB Cell and Tissue Staining Kit (Catalog # CTS019; brown) and nuclei were counterstained with hematoxylin (blue). B. Chromogenic detection by ICC was used to visualize IFN-gamma in human peripheral blood mononuclear cells stimulated with calcium ionomycin (0.5 µg/mL) and PMA (50 ng/mL) using anti-IFN-gamma affinity-purified polyclonal antibody (Catalog # AF285). Cells were stained using R&D Systems anti-goat HRP-DAB Cell and Tissue Staining Kit (Catalog # CTS008; brown) and nuclei were counterstained with hematoxylin (blue).
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