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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry
- Transfer 3-5 dorsal hippocampal sections for each mouse into 24 multi-well plates containing 500 µl of 0.1 M phosphate-buffered saline (PBS) for each well.
- Wash the sections contained in each well with 500 µl of 0.1 M PBS (5 min, 3 times with gentle shaking).
- Quench endogenous peroxidase activity by incubating sections in 1% hydrogen peroxide (H2O2) in PBS for 20 min with gentle agitation.
- Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
- Permeabilization of tissue: incubate sections for 5 min in 0.2% Triton X-100 in PBS.
- During the above steps (1.4; 1.5), make enough blocking buffer (10% Goat Serum and 0.1% Triton-X100 in PBS) for the entire experiment (number of sections x 100 µl x 3 steps).
- NOTE: Use serum from the same species in which the secondary antibody (Ab) (conjugated to biotin) was made.
- Prevent non-specific binding of the antibody by incubating sections in a blocking buffer (100 µl/section) for 1 hr at room temperature (RT) with gentle agitation.
- Incubate sections in primary Ab phosphorylated extracellular signal-regulated kinase (pERK) diluted in blocking buffer overnight at 4 °C, with gentle agitation.
- Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
- Incubate with the secondary Ab conjugated with biotin (1:250) diluted in blocking buffer (same as used in step 1.6) for 1 hr at RT with gentle agitation.
- Prepare the avidin-biotin complex (ABC) reagent at the same time, as avidin and biotin require at least 30 minutes at room temperature to complex.
- Prepare the required volume of ABC reagent according to the manufacturer’s protocol.
- Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
- Incubate sections for 45 min at RT with ABC Reagent.
- Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
- To prepare a diaminobenzidine (DAB) working solution, every step involving DAB must be done in the fume hood, as DAB is carcinogenic and teratogenic.
- Transfer the DAB solution to a new plate with a plastic transfer pipette.
- Place sections in the plate containing the DAB working solution and slowly swirl the plate by hand to allow maximum exposure to sections.
- Monitor DAB reaction on microscope until adequate signal develops (2-5 min).
- Stop the reaction at the desired color intensity by washing the tissue in 0.1 M PBS (3 x 5 min).
- Use bleach to deactivate any remaining DAB substrate solution in the fume hood overnight and dispose of it according to laboratory guidelines.
- Mount sections on gelatin-coated slides by floating them in PBS. Then, dry them at room temperature for at least 3-4 hours.
- Dehydrate the sections sequentially in 50%, 70%, 95%, and 100% ethanol for 2 min each and clear in 100% xylene for 5 min.
- Coverslip using mounting medium and leave in a fume hood to dry overnight.