Immunohistochemistry
Mice were anesthetised via intraperitoneal administration of
ketamine/xylazine (100mg/kg (Parnell)/xylazine ( 10mg/kg, Sigma
Cat. No. #X-1251) and cardiac perfused with ice cold PBS followed by
ice cold 4% paraformaldehyde (PFA) ( Scharlau, Cat. No.
PA00950500). Isolated brains were post-fixed in 5mL of 4% PFA overnight
at 4°C, then placed 30% sucrose solution (Chem-supply, Cat. No. SA030)
for 48 hours. The brains were then embedded in optimal cutting
temperature media (OCT) (Sakura, Cat. No. 4583) and coronally sectioned
at a thickness of 30µm. Sections were washed three times with PBS on a
rocker at 20RPM for 5 minutes each followed by 2-hour incubation in
blocking buffer containing 10% goat serum (Sigma-Aldrich, Cat. No.
G9023) and 1% Tween-20 (Sigma-Aldrich, Cat. No. P1379). Following
blocking, sections were rinsed in PBS followed by overnight incubation
with primary antibody (Supplementary Table 5) on a rocker at 20RPM at
4°C. Sections were then rinsed with PBS and incubated at room
temperature for one hour with secondary antibodies (Supplementary Table
6). Sections were again rinsed three times with PBS followed by one
rinse with milliQ H20 before being mounted onto
Superfrost plus™ slides ( Thermo Scientific, Cat. No. SF41296SP)
with Vectashield HardSet antifade mounting medium with DAPI (Abacus ALS,
Cat. No. H-1500). Slides were imaged using a Zeiss Observer Z1 widefield
microscope and Zen 2.6 (Zeiss, blue edition) software.