Immunohistochemistry Walkthrough

This guide is an overview of the main considerations for IHC, with hints and tips along the way. We've broken it down into stages.

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Tissue Fixation and Embedding

Tissue preservation is crucial to preserve antigens. There are several methods with a number of factors influencing which is the most appropriate for your experiment.

Fixation

Slide Preparation

Keeping your tissues, solutions, and slide labels on.

Slide Preparation

Antigen Unmasking

A must have step for many antibodies used on FFPE. As shown above, the difference without & with! Also known as epitope retrieval and by acronyms PIER, HIER, & HTAU.

Antigen Retrieval

Blocking Steps

What is in your tissue that could give erroneous results, as in the image above, and how do you tackle it?

Blocking Steps

Choosing a Primary Antibody

The primary antibody is the key reagent in your test. There's a number of things to consider for success.

Choosing a Primary

Primary Optimisation

Standardising IHC detection is most practical to control variables, so it's most common to optimisethe primary to fit in the standard protocol.

Optimising a Primary

Detection Systems

If you need an overview of detection systems here's our brief review. Skip to ABC detection or Polymer detection if you know which you prefer!

Detection Methods

ABC Detection

ABC Detection kits neatly balance sensitivity and flexibility for resarchers who work with a wide range of species.

ABC Kits

Polymer Systems

Polymer systems offer a shorter method than ABC kits with similar sensitivity. Usually provided ready to use, these kits require minimal optimisation and maximum consistency, perfect for very busy labs, or those who want the best results with the least effort.

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Enzyme Substrates

IHC has a reputation for being 'brown' but it doesn't have to be that way! Alternative colours can also be useful to contrast with pigmented tissues. Sensitivity and other charateristics can be important when planning your experiment.

Enzyme Substrates

Counterstains

A light counterstain is often employed to elucidate tissue morphology to aid identification. Hematoxylin is often readily available in histology labs, but there are other choices.

Counterstains

Mounting Sections

The final step for IHC is to mount the sections to preserve them, but a misstep here can leave you watching the staining disappear before your eyes, here's what to look out for.

Mounting Media