Slowfade gold with dapi mounting media

The fluorescent in situ assay shown in Figures 5C, 5E, 6I, S4B, S4C, and S7G–S7I was performed using the RNAscope 2.5 HD—multiplex fluorescent Manual Assay kit (ACDbio Cat. 320850). In this assay tissue was prepared and pretreated in the same way as in the chromogenic assay (see above). After incubation with target probes, 3 amplification steps and 1 detection step were performed following manufacturer instructions. Slides were mounted using the SlowFade Gold with DAPI mounting media (Life Tech Cat. S36939) covered with 1.5 glass coverslip (Fisher Cat. 12544E) and sealed with clear nail polish. Slides were imaged using the Axio Imager.Z2 fluorescent microscope (Zeiss). A 16 bit 1388 X 1040 pixel images were acquired using AxioCam HR3 camera (Zeiss). Fluorescent signal was imaged at 20x magnification using the Apotome.2 module (Zeiss). Z stack images (optical slice 1 μm) were obtained for each image. Set exposure was used when comparing signal between WT and KO mice for a given experiment. For cell type specific expression quantification experiments shown in Figures 5C–5E slides were imaged using the Zeiss LSM 710 confocal microscope at 20X magnification. 16 bit images at 0.16 μm/pixel were generated. A z stack (optical slice 1 μm, 6 slices total) was obtained for each image. Example images show maximal intensity projections for each group.

The slides containing the sections were blocked for 1 hr at room temperature in blocking buffer containing antibody buffer (100 mM L-lysine and 0.3% Triton X-100 in PBS) supplemented with 10% heat-inactivated normal goat serum. Primary antibodies diluted in antibody buffer with 5% goat serum were incubated overnight at 4°C. The next day, slides were washed 3 × 5 min with PBS with 0.2% Triton X-100 and secondary antibodies conjugated to Alexa Fluor (Molecular Probes) were applied for 2 hr at room temperature. Slides were mounted with the SlowFade Gold with DAPI mounting media (LifeTech #S36939), covered with 1.5 glass coverslip (Fisher #12544E) and sealed with clear nail polish. The following antibodies were used: Chk anti-GFP (Millipore #06-896, 1:500), Rb anti-SOX9 (Abcam #ab185966, 1:2000), Rb anti-ALDH1L1 (Abcam #ab-87117, 1:500), Rb anti-HA (CST #3724), Rb anti-S100β (Abcam #ab52642, 1:100), Ms anti-NEUN (Millipore #MAB377 1:100), Rb anti-NG2 (Millipore # Ab5320), Rb anti-MOG (Proteintech # 12690-1-ap), Rb anti-IBA1 (Wako #016-20001), Gp anti-VGLUT1 (Millipore #AB5905, 1:2000), Gp anti-VGLUT2 (Millipore #AB2251 1:3000, 1:5000), Rb anti-GLUA1 (Millipore #AB1504, 1:400), Rb anti-GLUA2 (Millipore #AB1768-I, 1:400), and Ms anti-Bassoon (Enzo #VAMP500, 1:500). All secondary antibodies were applied at 1:500 dilution.
The following mouse lines and antibody combinations were used: