Immunostaining for ATRX in Mouse Brains

Mice were perfused and the brains fixed for 72 h with 4% paraformaldehyde in PBS and cryopreserved in 30% sucrose/PBS. Brains were flash frozen in Cryomatrix (Thermo Fisher Scientific) and sectioned as described previously (Ritchie et al., 2014 (link)). For immunostaining, antigen retrieval was performed by incubating slides in 10 mM sodium citrate at 95°C for 10 min. Cooled slides were washed and incubated overnight in anti-ATRX rabbit polyclonal antibody (Santa Cruz Biotechnology, SC-15408; 1:200, H-300) (Watson et al., 2013 (link)) diluted in 0.3% Triton-X100 in PBS. Sections were washed and incubated with goat anti-rabbit Alexa Fluor 594 (Life Technologies) for 1 h. Sections were counterstained with DAPI and mounted with SlowFade Gold (Invitrogen). Cell counts were done in three control–KO littermate-matched pairs in a blinded manner.