IHC: Staining Protocol - Slide Mounted

This protocol is suitable for the immunohistochemical analysis of formaldehyde or glyoxal fixed cryo-tissue-sections. The tissue sections are stained on slides. For tissue preparation please refer to our tissue preparation protocol - formaldehyde or tissue preparation protocol - glyoxal.

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

Materials and reagents

TBS: Tris buffered saline (50 mM Tris, 150 mM NaCl), pH 7.2
Blocking buffer: 10% normal serum, 0.3% Triton X-100 in TBS (if secondary antibodies are used, normal serum from the
host-species of the secondary antibodies is recommended for blocking) (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 (if secondary antibodies are used, normal serum from the
host-species of the secondary antibodies is recommended for blocking)(normal serum from the host-species of the secondary antibodies is recommended for blocking).
Antibody and the corresponding species-specific secondary antibody
Optional: DAPI nuclear stain
Hydrophobic barrier pen
Mounting medium

Procedure

Take cryo-tissue sections from -20°C.
Optional: Perform antigen retrieval (AGR).
Air-dry sections at room temperature (RT).
Surround tissue with hydrophobic pen.
Rehydrate sections for 10 min in TBS at RT in staining dishes.
Add blocking buffer and block for 1 h at RT in a wet chamber.
Remove blocking buffer and add incubation buffer with the primary antibody.
Incubate primary antibody overnight at 4°C in a wet chamber. Important: Some antibodies require incubation at RT. Please refer to the corresponding antibody data sheet.
Wash slides three times for 10 min in TBS at RT in staining dishes.
Transfer the slides back to the wet chamber and apply the incubation buffer with the secondary antibody diluted to the manufacturer's recommended concentration.
Incubate for 1 h at RT. Important Notes:
- 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.
- Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye.
Wash slides three times for 10 min in TBS at RT in staining dishes RT (orbital shaker: 70 - 80 rpm).
Optional: Add DAPI solution for 10 min in TBS at RT.
Wash slides twice for 10 min in TBS at RT in staining dishes.
Wash slides with tap water.
Remove the hydrophobic circle around the tissue section.
Mount slides.

Note: This protocol has been validated in the SYSY Antibodies laboratories to ensure consistent and reliable staining results.
However, for achieving the best specific signal with minimal background, the optimal antibody concentration, incubation
temperature, and incubation duration should be optimized for each experiment.