Posted on November 8, 2021
An Overview Of IHC Staining Procedures For FFPE Tissues
Fixation is the most critical step in any histology procedure, including IHC. Tissues are typically fixed in 10% Neutral Buffered Formalin, and then processed and embedded into paraffin, formalin, paraffin-embedded, (FFPE) tissues. It is not critical to the IHC that tissues remain fixative for routine H&E or special staining procedures. However, there are so many repositories available online. You can click this link to find one that performs FFPE block storage with full care and attention.
However, it is crucial for IHC. Cross-linking in tissues' proteins can cause formalin to become a part of the protein. These cross-links can obscure epitopes that are needed to bind to IHC antibodies. Tissues that are too fixed can make them unusable for IHC. Tissues for IHC should be left to fix for at least 24 hours before being transferred to 70% alcohol for processing, embedding, and sectioning.
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Over-fixation can cause irreversible cross-linking of proteins within tissues. However, the optimal formalin fixation will create cross-links. Antigen (epitope), retrieval can remove these cross-links. Two of the most popular methods for antigen retrieval are heat and enzymes. HIER (heat-induced epitope retrieval) involves heating the slides to temperatures between 90 and 125 degrees Celsius for at least an hour.
The HIER process involves placing deparaffinized, hydrated slides in a buffered solution. This buffered solution is then heated in a pressure cooker or microwave. EIER is generally done on hydrated tissue sections for between 5 and 30 minutes at either 37 degrees Celsius or room temperature.
After the retrieval procedures have been completed, tissues are blocked for any endogenous tissue elements that could interfere with staining. Endogenous elements include proteins, avidins, biotin, and peroxidase. After blocking has been completed, the antibody can be applied to the tissue.