IHC on Frozen Tissue

Our protocol for performing IHC on frozen tissue sections.

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Immunohistochemistry On Frozen Tissue

Unlike paraffin samples, frozen samples are not treated with a fixative, so the antigens are not cross-linked with other proteins and therefore do not require an antigen retrieval step to unmask them for recognition by antibodies.

For fast processing of clinical samples, eliminating the fixation step by directly freezing and embedding with OCT, followed by cutting (6-8 μm thickness), is recommended as it would be time-saving and avoid the increased difficulty of sectioning caused by fixation. As for the temperature in the cryostat for unfixed tissues, please refer to the table below:

Cryostat Temperature For Unfixed Tissues
Brain, liver and lymph node tissues -10ºC/-15ºC
Thyroid, spleen, kidney and muscle tissues -15ºC/-20ºC
Tissue containing fat -25ºC
Tissue containing plenty of fat -30ºC
Tip:  Fresh tissue and fixation using 4% PFA in 4ºC overnight are recommended.
IHC on frozen tissue steps
  1. Wash tissue 3 times with PBS for 5 minutes each.

  2. Immerse tissue in 20–30% sucrose for 16–48 hours.

  3. Place the tissue block onto a pre-labeled tissue base mold.

  4. Cover the entire tissue block with cryo-embedding media (e.g., OCT).

  5. Slowly place the base mold containing the tissue block into liquid nitrogen until the entire tissue block is submerged into liquid nitrogen to ensure tissue is completely frozen.

  6. Store the frozen tissue block at -80ºC until ready for sectioning.

  7. Transfer the frozen tissue block to a cryotome cryostat (e.g., -20ºC) prior to sectioning and allow the temperature of the frozen tissue block to equilibrate to the temperature of the cryotome cryostat.

  8. Section the frozen tissue block to the desired thickness. Keep the section apparatus, including the blade and the blade holder, and clean by polishing with soft paper tissue. 

  9. Place the tissue sections onto glass slides suitable for immunohistochemistry. Sections can be stored in a sealed slide box at -80ºC for later use. Before further use equilibrate the tissue first at -20ºC for 30 minutes.