2.6. Immunohistochemistry
Mice (6-8 mice per strain) were perfused transcardially with 4% Paraformaldehyde (PFA) in Phosphate-buffered saline (PBS), brains were removed and post-fixed in the same solution for 24 hours at 4 °C. For cryoprotection, they were further incubated with 30% sucrose/PBS for 48 hours. Brains were then snap-frozen, cut in 30 µm thick horizontal sections in a Cryostat (Leica Biosystems) and slices were kept at 4°C in 0.02% sodium azide containing PBS until further processing. Slices were permeabilized and blocked in PBS containing 5% Donkey serum, 5% BSA and 0.3% TritonX for 1 hour. Rabbit anti-PV (1:500, ab11427, Abcam), rabbit anti-SST (1:250, sc13099, SantaCruz) and mouse anti-GAD67 (1:2500, MAB5406, Millipore) primary antibodies were used overnight at 4°C on corresponding slices. Slices were then incubated in donkey anti-rabbit Alexa-488 (1:1000, A21206, Invitrogen) or biotinylated goat anti-mouse (1:200, BA-9200, Vector Labs) secondary antibodies at room temperature for 2 hours. When biotinylated secondary antibody was used, antigen-antibody complexes were visualized by a final Strepdavidin-Cy5 (1:1000, Invitrogen) incubation for 30 min at room temperature. Finally, slices were incubated in 300 nM working solution of DAPI in PB for 5 minutes as a nuclear stain and mounted on glass slides using Immu-Mount (Thermo Fischer Scientific). Images were acquired with DMI6000 epifluorescence microscope (Leica Microsystems) using 10x objective and tile-scan function. Three to six slices per mouse were used for cell number analyses in ImageJ (NIH). Hippocampal subregions (CA3 and CA1) were traced based on DAPI signal and corresponding regions were analysed for signal intensity specific to antigen-antibody complexes. To calculate cell numbers, Cell Counter plug-in of ImageJ was used and counted cells were cross-checked using the DAPI channel. Finally, cell number was first divided to the area of the traced regions and final values were normalized against the same measures obtained from the B6J mice.