Mice were deeply anesthetized with isoflurane and transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (1× PBS). Brains were post-fixed for 1–3 days, washed in 1× PBS and sectioned (40 μm) coronally using a Vibratome (Leica). Slices were then immunostained for antibodies described. Slices were incubated in a 1× PBS blocking solution containing 5% normal horse serum and 0.3% Triton X-100 for 1 hour at room temperature. Slices were then incubated overnight at 4°C in the same solution containing primary antibodies at the following concentrations (1:100, NeuN; 1:500, Olig2; see above section “Cell class, type and subtype acquisition bias in Drop-seq datasets” for antibody details). The next morning, sections were washed three times for five minutes in 1× PBS for and then incubated for 1 hour at room temperature in the blocking solution containing donkey anti-goat Alexa 647 or Alexa 568. After drying, slices were mounted on slides (Super Frost) and allowed to dry. ProLong antifade mounting media containing DAPI (Molecular Probes) was applied and slides were coverslipped and sealed.
