Sample preparation for IHC
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Sample preparation is key to producing high quality staining during immunohistochemistry (IHC).
Sample preparation may include processes such as fixation, dehydration, embedding and sectioning. The two main methods of preserving tissues for IHC are paraffin embedding and freezing of the tissue (summarized below). The most appropriate route of sample preparation is usually determined by one or two experimental variables. For example, tissues may need to be snap-frozen if a phosphorylated epitope is being studied. Fixation of the tissue sample is performed to preserve tissue morphology and retain the antigenicity of the target protein during the IHC experiment. The method of fixation often drives the design of the sample preparation workflow. Embedding following dehydration is often used both to preserve tissue morphology and to give the tissue support during sectioning.
Additional steps in sample preparation for IHC may include antigen retrieval to unmask any epitopes that have been altered by fixation, permeabilization to grant the antibody access to intracellular proteins and blocking to prevent non-specific staining.
Tissue preservation guidelines
| Paraffin-embedded tissue | Frozen tissue | |
Fixation | Pre-embedding | Pre/post-sectioning |
| Sectioning | Microtome | Cryostat |
| Storage | Multiple years at room temperature (Note: antigen may change over time) | 1 year at -80°C (longer at -190°C) |
| Advantages | Preserves tissue morphology | Preserves enzyme function and antigenicity Shorter protocol (lengthy fixation step usually not required) |
| Limitations | Overfixation can mask the epitope | Formation of ice crystals may negatively affect tissue structure Sections produced are often thicker than paraffin sections, increasing potential for lower resolution and poorer images |
| Downstream protocols | DNA and RNA for PCR amplification (extensive crosslinking prevents extraction of long nucleotide strands, free nuclei for ploidy and cell cycle analysis, cells for flow cytometry) | DNA, RNA, free nuclei for FISH or cell cycle analysis |
| Precautions | Duration and intensity of tissue heating should be kept to a minimum as melting temperature of paraffin wax (50-60°C) can be deleterious to staining of some antigens | Tissues should be frozen rapidly to prevent formation of ice crystals and tissues should be allowed to reach cutting temperature (-20°C) in cryostat to avoid shattering As the tissues retain enzymatic activity, care should be taken to block the functions of endogenous enzymes that may affect the IHC detection method |
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