Prolong gold antifade media with dapi

Mice were anesthetized using a ketamine/xylazine/acetapromide cocktail and sacrificed by transcardial perfusion fixation with 15 mL ice cold 0.01 M PBS followed by 25 mL 4% paraformaldehyde (PFA) in 0.01 M PBS. Brains were post-fixed in 4% PFA overnight at 4°C and cryoprotected in 20% sucrose for 24 hrs at 4°C before being stored at −80°C until used for immunohistochemistry. Immunofluorescence histochemistry was performed as described below. Free-floating sections were cut at 30 µm from perfused brains using a sliding microtome (Leica SM2000R, Leica Microsystems, Wetzlar, Germany). Hypothalamic sections were collected from the division of the optic chiasm (bregma −1.0 mm) caudally through the mammillary bodies (bregma −3.0 mm). Sections were incubated for 30 min at room temperature in blocking reagent (5% normal donkey serum in 0.01 M PBS and 0.3% Triton X-100). After the initial blocking step, sections were incubated in rabbit anti-mouse Iba-1 (1:500, DAKO) in blocking reagent for 24 hrs at 4°C, followed by incubation in donkey anti-rabbit Alexa 555 (1:1000) for 2 hrs at room temperature. Between each stage, sections were washed thoroughly with 0.01 M PBS. Sections were mounted onto gelatin-coated slides and coverslipped using Prolong Gold Antifade media with DAPI (Thermofisher, Waltham, MA).