Freezing sliding microtome

Mice were anesthetized with sodium pentobarbital (40 mg/kg) before transcardial perfusion with PBS, immediately followed by 4% paraformaldehyde (PFA) in PBS, pH 7.4. Brains were post fixed overnight at 4°C before sequential 24 h incubations in PBS with 10%, 20%, and 30% sucrose. Brains were sectioned coronally at 40 μm using a freezing sliding microtome (Thermo Scientific). Sections were stored at 4°C in a cryopreservative solution (45% PBS, 30% ethylene glycol, 25% glycerol, by volume). For immunostaining, brain sections were rinsed several times with PBS before blocking with 5% normal goat serum and 0.02% Triton X-100 in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies, including rat anti-M2 AChR antibody (1:500, MAB367, Millipore) and rabbit GFP antibody (1:500, NB600-308, Novus), diluted in normal goat serum at 4°C overnight, and then rinsed several times with 0.02% Triton X-100 in PBS. Tissues were then incubated with secondary antibodies, including Alexa Fluor-488 goat anti-rabbit IgG (1:500, #A11008, Invitrogen) and Alexa Fluor-568 goat anti-rat IgG (1:500, #A11077, Invitrogen) for 1 hour. 4’,6-diamidino-2-phenylindole (DAPI, 7mg/mL, D1306, Invitrogen) was added during the secondary antibody incubation for nuclear counterstaining. Images were acquired with a Zeiss LSM 780 or 710 confocal microscope.