IgG/ChAT costaining.

Slides were thawed at RT, rehydrated in PBS for 3 hours, and dried overnight at 4°C. The next day, slides were permeabilized and blocked by incubation for 4 hours in 0.3% Triton X + 5% BSA (in PBS), then washed for 5 minutes in PBS. Slides were incubated with a rabbit anti-ChAT antibody (EPR 16590; Abcam) at 1:250 in the blocking solution for 48 hours at 4°C. Slides were then incubated with Alexa Fluor 594 goat anti-rabbit secondary antibody at 1:750 and with Alexa Fluor 488 goat anti-human IgG(H+L) at 1:500 in PBS for 2 hours at RT.

All slides were washed 3 times for 5 minutes at RT and dried before mounting with the DAPI-containing VectaMount Permanent Mounting Medium (Vector Laboratories), then sealed with nail varnish. Slides were stored at 4°C in the dark before imaging on Olympus FV1000 confocal microscope. All images across conditions were taken on the same day for a given staining. Microscopy images were processed minimally on FIJI.