Immunofluorescence Staining of Brain Sections

For Figs. 2, 3 and 6g, fixed brain sections were washed with PBS three times at room temperature (20–25 °C) prior to the procedure to remove the sucrose and freezing compound residue. The sections were then incubated with blocking solution (5% goat serum, 0.5% (v/v) Triton X-100, PBS) for 1 h at room temperature, then with the primary antibody and 4,6-diamidino-2-phenylindole (DAPI) diluted in blocking buffer for 16–20 h at 4 °C, washed in PBS three times for 30 min and incubated with secondary antibody diluted in the blocking buffer for up to 4 h at room temperature. Next, sections were incubated with PBS for 30 min three times and mounted with a cover glass (Menzel-Gläser) and Immu-Mount mounting medium (Shandon, Thermo Scientific). For experiments with dual mRNA and protein labeling, instead of mounting after the hybridization protocol, the sections were subjected to the IHC as described here. For IHC staining in Fig. 6e,f, the previously described procedure was used^{18 (link)}.