Imaging Hippocampal Neuronal Activation In Mouse Brain Sections

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Immunohistochemistry

1. 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.
2. Wash the sections contained in each well with 500 µl of 0.1 M PBS (5 min,  3 times with gentle shaking).
3. Quench endogenous peroxidase activity by incubating sections in 1% hydrogen peroxide (H₂O₂) in PBS for 20 min with gentle agitation.
4. Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
5. Permeabilization of tissue: incubate sections for 5 min in 0.2% Triton X-100 in PBS.
6. 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).
7. NOTE: Use serum from the same species in which the secondary antibody (Ab) (conjugated to biotin) was made.
8. 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.
9. Incubate sections in primary Ab phosphorylated extracellular signal-regulated kinase (pERK) diluted in blocking buffer overnight at 4 °C, with gentle agitation.
10. Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
11. 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.
12. 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.
13. Prepare the required volume of ABC reagent according to the manufacturer’s protocol.
14. Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
15. Incubate sections for 45 min at RT with ABC Reagent.
16. Wash sections in 0.1 M PBS with gentle agitation (3 x 5 min).
17. To prepare a diaminobenzidine (DAB) working solution, every step involving DAB must be done in the fume hood, as DAB is carcinogenic and teratogenic.
18. Transfer the DAB solution to a new plate with a plastic transfer pipette.
19. Place sections in the plate containing the DAB working solution and slowly swirl the plate by hand to allow maximum exposure to sections.
20. Monitor DAB reaction on microscope until adequate signal develops (2-5 min).
21. Stop the reaction at the desired color intensity by washing the tissue in 0.1 M PBS (3 x 5 min).
22. Use bleach to deactivate any remaining DAB substrate solution in the fume hood overnight and dispose of it according to laboratory guidelines.
23. Mount sections on gelatin-coated slides by floating them in PBS. Then, dry them at room temperature for at least 3-4 hours.
24. Dehydrate the sections sequentially in 50%, 70%, 95%, and 100% ethanol for 2 min each and clear in 100% xylene for 5 min.
25. Coverslip using mounting medium and leave in a fume hood to dry overnight.

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