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.