Goat anti pdgfrα

Mice were perfusion‐fixed with 4% paraformaldehyde (PFA; Sigma) (wt/vol) in phosphate buffered saline (PBS). Brains were cut into 2 mm‐thick coronal slices using a 1 mm brain matrix (Kent Scientific) before being post‐fixed in 4% (wt/vol) PFA at 21°C for 90 min. Tissue was cryoprotected in 20% sucrose (Sigma) in PBS and snap frozen in OCT (ThermoFisher) for storage at −80°C. 30 μm coronal brain cryosections were collected and processed as floating sections (as per O'Rourke et al., 2016). Primary and secondary antibodies were diluted in PBS blocking solution [0.1% (vol/vol) Triton X‐100 and 10% fetal calf serum in PBS] and applied to cryosections overnight at 4°C. Primary antibodies included goat anti‐PDGFRα (1:200; GeneTex, California), rabbit anti‐OLIG2 (1:400 Millipore) and rat anti‐GFP (1:2000; Nacalai Tesque, Kyoto, Japan). Secondary antibodies were conjugated to AlexaFluor‐488, −568 or − 647 (Invitrogen) and included: donkey anti‐goat (1:1,000), donkey anti‐rabbit (1:1,000), and donkey anti‐rat (1:500). Nuclei were labeled using Hoechst 33342 (1:1,000; Invitrogen).

Cryosections (30μm) were collected as floating sections from brain, spinal cord, eye, spleen, liver, kidney, heart, adrenal gland and pituitary gland. 30μm sections from sciatic nerve, gastrocnemius, intestine, and bone marrow (tail) were collected directly on to glass slides. Sections were processed for immunohistochemistry as previously described [24 (link)], using the following primary antibodies: goat anti-PDGFRα (1:200; GeneTex, California, USA), rat anti-GFP (1:2000; Nacalai Tesque, Kyoto, Japan; 04404–84), rabbit anti-S100β (1:500; Dako Australia Pty. Ltd., Campbellfield, Australia) and mouse anti-S100β (1:500; Sigma). Protein expression was visualised by secondary antibodies conjugated to Alexa Fluor-488, -568, -594 or -637 (Invitrogen): donkey anti-rat (1:500), donkey anti-goat (1:500), donkey anti-rabbit (1:500), donkey anti-mouse (1:500). Tissue was also exposed to Hoechst 33342 (Invitrogen; 1:10,000 dilution) to visualise the nuclei. Floating sections were mounted onto glass slides and the fluorescence preserved by the application of fluorescent mounting medium (Dako Australia Pty. Ltd., Campbellfield, Australia).