Publication protocol
Mice were anesthetized intraperitoneally with ketamine hydrochloride and xylazine (100 and 15 mg.kg-1, respectively). They were then perfused transcardially with 0.9% saline containing heparin (100–200 UI/ml followed by 30–50 ml of a fixative solution containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB). Dissected brains were post-fixed overnight in the same solution at 4°C, rinsed three times for 3 min, and then placed in a 30% sucrose solution at 4°C for at least 24 h. Horizontal sections of thickness 60 μm were cut in 0.1 M PBS using a slicing vibratome (Microm HM650 V). Membranes were permeabilized by three cycles of freeze-thawing slices on dry ice in a 30% sucrose containing solution. Sections were washed three times (2 × 30 min, 1 × 60 min) in PBS 0.1M (BupHTM Phosphate Buffered Saline Packs, Thermo Fisher Scientific), then transferred to a saturation buffer containing 2% milk powder and 0.3–0.4% Triton X-100 in PBS 0.1M, and agitated for 2 h at room temperature. Sections were then transferred into primary antibody solution of 0.1M PBS and 0.3% Triton X-100 and gently agitated, overnight at 4°C. Sections were rinsed three times (2 × 30 min, 1 × 60 min) in PBS then incubated in dilutions of secondary antibody, conjugated to different fluorophores, for 4 h at room temperature under gentle agitation. 4′,6-diamidino-2-phenylindole (DAPI, Sigma) was always added to secondary antibodies containing solutions (1:1000) to stain cellular nuclei. For SOM immunostaining, we increased the incubation time with the primary antibody to 48–72 h and with the secondary antibody to 24 h, both at 4°C. For SOM and PV co-labeling, these long incubation times were applied as well.
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