Immunofluorescence Imaging of Neuronal Markers

Immunofluorescence was performed as described previously (96 (link)). Briefly, mice were terminally anesthetized with 7% choral hydrate (500 mg⋅kg⁻¹; Sigma-Aldrich) diluted in saline and transcardially perfused with 0.1 M PBS followed by 10% neutral-buffered formalin solution (NBF) (Thermo Fisher Scientific). Brains were extracted and postfixed overnight at 4 °C in NBF, cryoprotected in 20% sucrose, and sectioned coronally at 30 µm on a freezing microtome (Leica Biosystems). The following primary antibodies were used overnight at room temperature: rabbit anti-HSD2 (H-145; Santa Cruz Biotechnology), 1:300; rat anti-mCherry (Life Technologies), 1:3,000. The following day, the sections were washed and incubated at room temperature in donkey Alexa Fluor fluorescent secondary antibody (Life Technologies; 1:1,000). Fluorescent images were captured using an Olympus VS120 slide-scanning microscope.