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IHC
IHC Embedding Optimization
Paraffin embedded vs. frozen tissue
Once your tissue sample is fixed using the appropriate method and fixative, it is ready for sectioning and mounting. In order to preserve tissue morphology during sectioning, the tissue must be embedded in paraffin. If you are performing IHC on frozen sections, fixation occurs after sectioning. The decision to use frozen or formalin fixed paraffin embedded (FFPE) samples is dependent on which downstream applications you intend to use.
| Praffin embedded | Frozen | |
|---|---|---|
| Fixation step | Pre-embedding | Post-sectioning |
| Sectioning equipment | Microtome | Cryostat |
| Stroage stability | Years at room temperature | One year at -20C |
| Advantages | Tissue morphology preservation | Enzyme activity preservation |
| Disadvantages | Causes more epitope masking | Ice crystals may form and disrupt tissue structure |
| Downstream applications | DNA/RNA PCR amplification | Free nuclei for FISH or cell cycle analysis |
| Preacautions | Temperature must be monitored to avoid melting wax before deparaffinization and mounting. | Tissues must be frozen and warmed slowly to avoid ice crystals and shattering, respectively. |
Keywords: IHC, optimization, ICH-P, IHC-F, FFPE, frozen section, paraffin embedding, paraffin embedded vs frozen
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