Kirrel3+Gad1 Fluorescent In Situ Hybridization (FISH) Data

Fluorescent in situ hybridization chain reaction was performed as described (Trivedi, Choi et al. 2018). Briefly, mouse brains were cryo-sectioned to 30 μm slices, mounted on slides, fixed in 4% paraformaldehyde (PFA), and washed in PBS. Slices were treated with 1 mg/ml proteinase K in TE buffer and equilibrated in SSC buffer. HCR probes were designed and generated by Molecular Instruments for Gad1, Kirrel3, and GFP. After nuclear staining with Hoechst in PBS, coverslips were mounted in Fluoromount-G (Southern Biotech catalog #0100-01) and imaged on a confocal microscope. For dual in situ/immunohistochemistry, samples were incubated in freshly prepared 4% PFA in 1xPBS made with DEPC water for 10 minutes and washed in 1X DEPC treated PBS (pH 7.4) for 5 minutes at room temperature three times. Samples were blocked for 1 hour at room temperature (100 μL/slide applied over the top of sections) inside a humidified chamber and 75 μL primary antibody solution (anti-mCherry 1:1000) was added per slide and covered with an RNAse free coverslip. Samples were incubated for 1 hour at room temperature and then overnight at 4°C in a humidified chamber. The next day, 8 samples were washed in 1X DEPC treated PBST (pH 7.4) for 5 minutes at room temperature for three washes before 2° antibody was added at 1:1000 then incubated for 1 hour at room temperature inside a humidified chamber. Samples were washed in 1X DEPC treated PBST (pH 7.4) for 5 minutes at room temperature three times and coverslipped with Fluoromount-G mounting reagent.