Preparing the Sample for an IHC Experiment

One of the most important precautions to ensure that staining during immunohistochemistry (IHC) is of good quality is proper preparation of the sample.

This includes various processing steps: fixing the tissue, dehydration, embedding and taking sections. Tissues intended for use in IHC are mainly preserved by either embedding in paraffin or by freezing. Which method is selected depends upon one or two of the other factors in the experimental conditions.

For instance, if a phosphorylated antigenic epitope is targeted, it may require snap-freezing. The tissue is fixed to keep the tissue morphology intact, as well as to make sure the target protein remains antigenic while IHC is being performed.

The procedure chosen for fixation is often the key to the rest of the workflow for sample preparation. Dehydration with subsequent embedding is used in many cases, as it retains the tissue morphology unchanged, and also supports the tissue while thin sections are being taken.

Sometimes, the IHC sample may need more steps than this, such as antigen retrieval to uncover any epitopes which underwent change during the process of fixation; permeabilization of the cell membrane so that the antibody can enter and bind to intracellular proteins; and blocking, which is intended to avoid the occurrence of non-specific staining.

Guidelines for Tissue Preservation

  Tissues embedded in paraffin Frozen tissue
Fixation Before embedding Either before or after sectioning
Sectioning With a microtome Using a cryostat
Storage Can be stored for a number of years at room temperature, but the antigen may show change with the course of time Can be stored for 1 year at -80 oC, but for longer at -90 oC
Benefits Tissue morphology unchanged Enzyme function intact, preserves antigen properties Avoids the need for fixation (a long procedure) and therefore, cuts down the length of the protocol
Issues If excessive fixation is carried out, the epitope may be masked Ice crystals can damage the structure of the tissue Sectioning is typically coarser than with paraffin-embedded sections, which leads to higher chances of decreased resolution and low-quality images
Downstream protocols Yields DNA and RNA PCR amplification, as the occurrence of much crosslinking prevents the extraction of long strands of nucleotides, free nuclei (required for ploidy and for cell cycle analysis), or cells (for flow cytometry analysis) Yields DNA, RNA, free nuclei suitable for FISH or cell cycle analysis
Precautions The tissue should be heated as little and for as short a time as is possible, as paraffin wax melts at 50-60 oC, which can prevent some antigens from being stained Rapid freezing is essential to avoid ice crystal formation. Tissues that are frozen should be cut only after they have attained cutting temperature of -20 oC in the cryostat, as otherwise they will shatter.
The presence of intact enzymes in the frozen tissue requires care to inhibit their functions, as otherwise the activity of these endogenous enzymes can affect the results of the IHC experiment

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Last updated: May 24, 2019 at 7:36 AM

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