Fluoromount aqueous mounting medium

Detection of Nlgn2, Nefh and Calca transcripts was performed by fluorescence in situ hybridization. Adult (>8-week-old) animals were killed by asphyxiation followed by cervical dislocation, and individual DRG were rapidly dissected from mice, frozen in dry ice-cooled 2-methylbutane and stored at –80 °C until further processing. The DRG were cryosectioned at a thickness of 20 µm, and RNA was detected using the RNAscope Fluorescent Multiplex Assay (ACD Bio), according to the manufacturer’s protocol. The following probes were used: Mm-Nlgn2 exons 3–5 (made to order, 300031), Mm-Nefh (443671) and Mm-Mrgprd (417921). Sections were mounted in Fluoromount Aqueous Mounting Medium (Thermo Fisher, 00-4958-02) and imaged on a Zeiss LSM 700 confocal microscope.

Cells were fixed with 4% paraformaldehyde (PFA; P6148, Sigma-Aldrich) for 20 min at room temperature (RT). Blocking was performed with blocking solution: 10% Fetal Bovine Serum (FBS) or 5% Goat serum (GS) with 0.2% Triton X 100 in PBS for permeabilization. Cells were then incubated with primary antibodies (anti-SSEA4, CST-4755, Cell Signaling Technology, Danvers, MA, USA; anti-TRA-1-60, ab16288, Abcam, Cambridge, UK; anti-OCT4, sc-5279, Santa-Cruz, Starr County, TX, USA; anti-FMRP, BLG-834601, Biolegend, San Diego, CA, USA; anti-Tuj1, BLG-801201, Biolegend; anti-PAX6, BLG-901301, Biolegend; anti-MAP2, sc-20172, Santa Cruz; anti-SYN1, AB1543, Merck, Darmstadt, Germany; anti-PSD-95, MAB1596, Merck) diluted in blocking solution for 1 h at RT, washed 3 times with PBS, and incubated with secondary antibodies (donkey anti-mouse Alexa Fluor 488, A21202, Thermo Fisher Scientific, Waltham, MA, USA; goat anti-rabbit Alexa Fluor 594, A11012, Thermo Fisher) for another 1 h at RT in the dark, and counterstained with DAPI for nucleus localization (D1306, Thermo Fisher Scientific). Cells were mounted with Fluoromount aqueous mounting medium (00-4958-02, Thermo Fisher Scientific). Bright-field, phase, and fluorescence images of cells were obtained using an Olympus IX51 inverted light microscope (Olympus, Tokyo, Japan).

Cells grown on Geltrex-coated 13 mm glass cover slips were fixed with 4% paraformaldehyde (PFA; Cat No. P6148, Sigma-Aldrich, St. Louis, MO, USA) for 20 min at room temperature (RT). Blocking was performed with blocking solution, 10% FBS with 0.2% Triton X 100 in PBS for permeabilization. Cells were then incubated with primary antibody diluted in blocking solution for 1 h at RT, washed 3 times with PBS and incubated with secondary antibodies for another hour at RT; they were then and counterstained with DAPI for nucleus localization (Cat No. D1306, Thermo Fisher Scientific). Cells were mounted with Fluoromount aqueous mounting medium (Cat No. 00-4958-02, Thermo Fisher Scientific). The antibodies used for the immunostaining are listed in Table 1. Bright-field, phase, and fluorescence images of cells were obtained using an Olympus IX51 inverted light microscope (Japan).