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Standard Protocol for Immunohistochemistry

This protocol is suitable for the immunohistochemical analysis of PFA perfused and post-fixed vibratome and cryo tissue-sections. For tissue preparation, please refer to our tissue preparation protocols.

Important: For good detection some proteins have special tissue preparation or antigen retrieval (AGR) requirements. Please refer to the remarks section for IHC on the respective data sheet.

Solutions needed:

  • TBS: 20 mM Tris, pH 7.2, 150 mM NaCl
  • Blocking buffer: 10% normal serum, 0.3% Triton-X 100 in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking)
  • Incubation buffer: 5% normal serum, 0.3% Triton-X 100 in TBS (normal serum from the host-species of the secondary antibodies is recommended for blocking)
  • Mounting medium
  • Optional: DAPI nuclear stain

Procedure:

  1. Transfer the free-floating sections into a staining dish containing TBS.
  2. Optional: Perform antigen retrieval (AGR).
  3. Transfer the sections to blocking solution and block for 1 h at RT (orbital shaker: 70 - 80 rpm).
  4. Transfer the sections to the incubation buffer with the primary antibody and incubate overnight at 4°C (orbital shaker: 60 rpm; optimal dilution must be determined experimentally). Important: Some antibodies require incubation at room temperature. Please refer to the corresponding antibody data sheet. 
  5. Wash three times for 10 min in TBS (RT; orbital shaker: 70 - 80 rpm).
  6. Transfer the sections to the incubation buffer with the secondary antibody diluted to the manufacturer's recommended concentration and incubate for 1 h at RT (orbital shaker: 70 - 80 rpm). Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye. Important Note: This step can be omitted when fluorophore conjugated primary antibodies are used. In Multiplex staining make sure to use secondary antibodies cross-adsorbed against the host species of the other primary antibody used in your experiment. Ideally all secondary antibodies should come from the same host species. If not, make sure that they have been cross-adsorbed against IgGs of the host-species of the other secondary antibody as well. This avoids cross-reaction between the secondary antibodies.
  7. Wash three times for 10 min in TBS (RT; orbital shaker: 70 - 80 rpm). Optional: Add DAPI to the first TBS washing step.
  8. Wash sections with tap water.
  9. Mount slides and microscope.

Note: The SYSY standard protocol generates good staining results in the SYSY labs and may be used as a reference. However, to achieve the highest specific signal and lowest non-specific background signal, the best antibody concentration, incubation temperature and incubation time for each antibody must be individually determined.