Immunofluorescence Staining of Frozen Tissue Sections

The frozen tissues were mounted on the specimen stages in the same way as described for “in situ hybridization”. Sections with 12 µm thickness were mounted onto a poly-l-lysine coated slide. Sections were fixed in cold 4% paraformaldehyde for 10 min after air drying. Slides were then processed for IF staining. IF was performed as previously described^{2 (link),4 (link)}. Frozen sections (12 µm) from each genotype were processed onto the same slides and incubated with primary antibodies listed in Supplementary Table 2. For signal detection, secondary antibodies listed in Supplementary Table 2 were used. Nuclear staining was performed using Hoechst 33342 (5 µg/mL, H1399, Thermo Scientific). Pictures were taken using the Nikon Eclipse 90i upright microscope and processed by Nikon Elements Viewer.