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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.
2BScientific Limited are pleased to offer in house support of this method. Why not give our experts a call to chat through your experiment, or for help troubleshooting?

Tissue Fixation and Embedding

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.

Slide Preparation

Slide Preparation

Immunohistochemistry can be a long protocol, so careful preparation of sections is required to ensure they can withstand the treatment they receive.

Antigen Unmasking

Antigen Unmasking

Also known as epitope retrieval, antigen unmasking helps make your target available to the primary antigen to bind, particularly when formalin fixation has caused conformational changes in the protein structure.

Blocking Steps

Blocking Steps

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

Choosing a Primary Antibodies

Choosing a Primary Antibodies

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

Optimising a Primary Antibody

Optimising a Primary Antibody

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

Detection Systems

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!

Enzyme Substrates

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 characteristics can be important when planning your experiment.

Counterstains

Counterstains

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

Mounting IHC Sections

Mounting IHC 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.

Dehydration / Rehydration in Immunostaining

Dehydration / Rehydration in Immunostaining

Some histology preparations rely on removal of water from sections to aid preservation.

Tyramide Amplification

Tyramide Amplification

A really powerful amplification tool – when handled with care!