M205 fa upright stereomicroscope

For the measurement of the infarct area, metabolically active tissue was stained with triphenyltetrazolium chloride (TTC). After frozen on dry ice for 2 hr, the harvested hearts were cut into 1 mm thick sections perpendicular to the long axis, as shown below. The sections were incubated in 1% w/v TTC (T8877, Sigma) prepared in 0.1 mol/L phosphate buffer pH 7.4 for 15 mins at 37° C. Following, the heart sections were fixed in 4% PFA for 15 mins before imaging with a Leica M205 FA upright Stereomicroscope. The images were analyzed using ImageJ. Infarct size was calculated by measuring white areas (infarct region) against the total area.

Brains were coronally cryosectioned (14 μm). Sections were taken throughout the whole hypothalamus (−0.46 to −2.80 mm caudal to bregma [21] ). Sections were fixed in 4% PFA, permeabilized in 0.25% Triton-X, and blocked with 10% SeaBlock (Calbiochem). For anti-HA immunohistochemistry, sections were incubated with rabbit polyclonal anti-HA (ab9110, Abcam) primary antibody (1:1000) followed by donkey anti-rabbit Alexafluor 488 (1:150) secondary antibody. Endogenous tissue autofluorescence was dampened by incubation with 0.15% Sudan Black. Slides were mounted with Prolong Gold Antifade Mountant with DAPI (Thermo Scientific). Images were collected on a M205 FA Upright Stereomicroscope (Leica) and captured using a DFC 565FX (Leica) camera through LAS AF v3.1.0.8587 software (Leica). Images were processed and analyzed using Image J.