IHC Protocol for Frozen and Paraffin Sections

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1. Paraffin vs Frozen Section

IHC protocol for frozen and paraffin sections is specified below.

1.1. IHC Protocol for Frozen Sections

Frozen section is usually applied in rapid pathological diagnosis during the surgery.

Advantages:

  • Keep antigen immune activity of the tissue better;
  • Antigen retrieval is not required for immunohistochemistry.

Disadvantages:

  • Ice crystal can be easily formed to destroy cell structure and may disperse antigen;
  • Thicker than paraffin section and looks unsightly.

1.2. IHC Protocol for Paraffin Sections

Paraffin section is widely used for routine slicing.

Advantages:

  • Maintain the cellular morphological structure of the tissue;
  • Stored for a long time at room temperature

Disadvantages:

  • Complex steps;
  • Antigen retrieval required

In applications of antibodies, frozen section tissue may be unsuitable for paraffin section. Baking at high temperature is required for paraffin section, which may destroy antigenicity of the tissue. The tissue with stable antigenicity is suitable for paraffin section. However, paraffin section tissue can be also processed as frozen section.

2. Dewaxing in Tissue Processing

Wax is insoluble in water. The residual wax on the section may result in uneven staining and increased background staining. Thus, the section must be completely dewaxed. Dimethylbenzene is the key reagent with strong dewaxing ability for less processing time.

Dewaxing time should be flexibly adjusted. In summer, the high room temperature requires 3-5 min. In winter, the low room temperature requires 10-20 min or longer.

The cut section can be stained after baking for 2h. The baked section can accelerate the dewaxing process. The cut and baked section still requires for heating 10-20 min first. Then, dewaxing is more rapid. In principle, completely dewaxing is essential.

3. Triton x 100 Permeabilization Protocol

Triton X-100 is a kind of detergent and surfactant with antioxidation. In immunohistochemistry (section>10μm) and immunocytochemistry, Triton X-100 functions as the cell permeabilization buffer to punch holes in the membrane.

Mechanism: Triton X-100 can dissolve lipids on cell membrane, nuclei membrane and organelle membrane. Then, antibodies and substances with macromolecule structure can enter cytoplasm and nucleus. It's strongly recommended to use Triton X-100 during cellular immunohistochemistry. Thus, antibodies can enter cell and bind with relevant antigens.

4. Recommended Antibodies

REFERENCES

A Single Simple Procedure for Dewaxing, Hydration and Heat-Induced Epitope Retrieval (HIER) for Immunohistochemistry in Formalin-Fixed Paraffin-Embedded Tissue, PMID: 26708177.