Rats were euthanized with lethal dosage of sodium pentobarbital (65 mg/kg, IP) and transcardially perfused with ice cold ~250 mL PBS followed by cold ~300 mL 4% paraformaldehyde (PFA). Brains were post-fixed in 4% PFA at 4° C for at least 16 hours (up to 24 hours). The brains were cryoprotected in 30% sucrose/PBS at 4° C until used. Brains were frozen with OCT compound (VWR Clear Frozen Section Compound, VWR, Mississauga, ON, Canada) in a Peel-A-Way embedded mold (PolySciences Inc., Warrington, PA, USA). Twenty μm coronal brain cryosections were cut and mounted onto Superfrost Plus slides and stored at −80° C. The localization of the PVN was determined according to the rat atlas of Paxinos and Watson [38 ].
For immunostaining, sections were first dried in the fumehood, then washed with PBS, and blocked in 5% goat serum in 0.2% Triton-100/PBS for 1 hour. Sections were then incubated in microglia-specific rabbit anti Iba-1 primary antibody (1:800, Wako Chemicals USA, Inc. Richmond, VA, USA) at 4° C overnight, followed by incubation with Alexa Fluor 488-labelled goat anti-rabbit IgG secondary antibody (1:500, Invitrogen, Rockford, IL, USA) in 5% goat serum in 0.2% Triton X-100/PBS. Sections were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA).
For immunostaining, sections were first dried in the fumehood, then washed with PBS, and blocked in 5% goat serum in 0.2% Triton-100/PBS for 1 hour. Sections were then incubated in microglia-specific rabbit anti Iba-1 primary antibody (1:800, Wako Chemicals USA, Inc. Richmond, VA, USA) at 4° C overnight, followed by incubation with Alexa Fluor 488-labelled goat anti-rabbit IgG secondary antibody (1:500, Invitrogen, Rockford, IL, USA) in 5% goat serum in 0.2% Triton X-100/PBS. Sections were mounted using Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA).