Inactivation and Blocking

Inactivation

When either the horseradish peroxidase (HRP) or alkaline-phosphatase (AP) system is applied for IHC, activation of endogenous enzymes should be blocked or inhibited to avoid producing non-specific binding.

  • Endogenous HRP Inactivation
    • Incubate the paraffin embedded section in 3% H2O2 for 10 min
    • Incubate the frozen section or cell section in solution composed of methanol and 3% H2O2 (v/v: 4:1) for 30 min
  • Endogenous AP Inactivation
    • Incubate the sample section in 0.1 mM Levamisole
    • Note: Levamisole cannot inhibit the AP activation of endogenous intestine tissue
  • Blocking
    Residual sites on the tissue section may bind to secondary antibody and produce follow-up false positive results. Therefore, serum from the same species as the secondary antibody is commonly used for blocking. Animal’s autoantibody in the serum can bind to the sites in advance. Blocking should be done at room temperature for 10-30 min (avoid excessive blocking).

Tissue Section Types

  • Frozen
    The most important feature for this type of tissue section is to keep antigen’s immune-competence completely, especially for the cell surface antigen. Both fresh and fixed tissues can be processed as frozen tissues. However, the tissues must be dried (or primary fixed) and stored at low temperature.
  • Paraffin-Embedded
    Paraffin-embedded tissue section is normally sliced by a rotary microtome to give a thickness of 2-7 μm. With proper treatment, the section reveals clear tissue structure and exact antigen location to enable high medical-value pathology researches and retrospective studies. This section type can be stored at 4℃ for long term use.

Processing

  • Cell on Coverslip
    • Place settled coverslip in culture bottle or perforated plate
    • Take out coverslip after cell growth has reached 60%
    • Fix coverslip with cold acetone or 4% paraformaldehyde for 10 to 30 min
    • Store cell (in gelatin) at -20℃
  • Cell Smear
    • Collect non-adherent cells and wash 2X by cold PBS buffer
    • Re-suspend cells with PBS buffer
    • Add 30-50 µL to settled slide and smear it evenly
    • Air dry slide a little bit and cover cell with 4% paraformaldehyde for 2-4 hours
    • Store cell (in gelatin) at -20℃