Immunohistochemistry Workflow

• Maximize tissue section retention and adherence on glass slides.
• Demarcate and isolate individual sections to reduce reagent use or for discrete treatment.

Increase tissue antigenicity ("immunoreactivity") in aldehyde-fixed preparations with defined pH and salt solutions. The combination of aldehyde fixation (e.g., formalin) and heat exposure from the paraffin-embedding process can make epitopes inaccessible to detection. During antigen retrieval, tissues are treated with a combination of buffered solutions and high temperature, which causes conformational changes in the proteins that expose antigens for detection.

Reduce or eliminate unwanted background (non-specific) staining on tissue sections and cell preparations using a blocking solution. Non-specific staining can result from endogenous enzyme or tissue elements, including endogenous enzyme activity, presence of Fc receptors, or interactions of detection reagents with tissue or cell proteins and other macromolecules. Choose a blocking solution based on the results of negative control sections.

Identify and localize the target protein antigen or glycoprotein moiety in cells or tissue preparations using specific validated markers. When choosing a primary antibody or lectin, consider the tissue species and tissue preparation method (e.g., fixation) to ensure specific binding to your target.

Select highly purified and optimally conjugated detection reagents to meet assay conditions, such as primary antibody species, tissue species, target abundance, and ease of use (concentrated or ready-to-use formats).

If needed, increase the sensitivity of detection to visualize weakly or transiently expressed, upregulated, or unknown (e.g., gene knock-in studies) antigens.

Visualize your target protein antigen and its cellular and/or extracellular localization and relative expression levels using enzyme-specific chromogenic color development. Choose an enzyme substrate that matches your color preference and that is compatible with other system reagents (e.g., counterstains, mounting media, and other substrates if multiplexing).

Use a contrasting nuclear stain to clarify the target antigen signal within heterogeneous morphological structures. This helps elucidate cellular architecture and specific staining patterns in tissues. Choose a counterstain (blue, green, or red) that contrasts with your enzyme substrate and that is chemically compatible with both the substrate and mounting medium.

Preserve the target antigen stain for short-term or indefinite storage and archiving. Choose a mounting medium that is compatible with both your substrate and your counterstain. Use a non-aqueous mounting medium to mount slides to be archived, or an aqueous mounting medium for storage up to a few years.