Immunohistochemistry Protocol - Mouse Antibodies
(4% Paraformaldehyde- & Formalin-Fixed Paraffin Embedded Tissue)
- Dewax sections through 3-4 changes of xylene and rehydrate through descending alcohol to water.
- Soak in PBS for 5 min.
- Quench endogenous peroxidase for 15 min at room temperature with 1.5% hydrogen peroxide in methanol.
- Antigen recovery/retrieval, (required for any nuclear antigen): Immerse sections in 10 mM sodium citrate, pH 6.0, and heat in a microwave for 15 min at 90oC.
- Blocking: For any monoclonal antibody use on mouse tissues, block using a MOM kit (Vector labs). For use on human tissue, use 2% normal horse serum in PBS + 0.2% Triton X-100 (normal serum block) for 2 hr at room temperature to block.
Staining procedure (for human tissue):
- Incubate samples overnight at 4oC with the primary antibody diluted in normal serum block (2-4% normal horse serum in PBS/0.2% Triton X-100) at the dilution indicated.
- Wash sections 5 times for 5 min each in PBS containing 0.2% Triton X-100.
- Incubate 30 min at room temperature with biotinylated horse anti-mouse antibody diluted 1:200 in blocking solution.
- To detect antigen:antibody complexes we recommend using a Vectastain ABC Peroxidase Elite Mouse IgG kit as follows (or use your own detection system):
- Incubate sections for 30 min at room temperature with avidin/biotin/peroxidase complex (ABC kit from Vectastain) diluted in blocking solution.
- Incubate with NiDAB in 0.1 M acetate buffer for 4 min at RT.
- Incubate with Tris cobalt for 4 min at RT.
- Counterstain with nuclear fast red for 2 min at RT.
Staining procedure (for mouse tissue):Follow directions for the Vector Labs MOM (Mouse On Mouse) kit.
- mouse IgG or nonimmmune sera at the same dilution as the primary antibody
- omission of the primary antibody to check for endogenous biotin and peroxidase activity, as well as nonspecific binding of the secondary antibody.