Prolong anti fade media

Hippocampal neurons were fixed with 4% paraformaldehyde in PBS containing 10% sucrose for 20 min at room temperature. After washing, neurons were permeabilized and blocked simultaneously in a solution containing 2% goat serum, 3% BSA and 0.2% Triton X-100 in PBS for 1 h at room temperature. The primary antibodies, anti-SESTD1 (1:1000), anti-PSD-95 (1:3000) or anti-bassoon (1:2000), were added in PBS containing 3% BSA and incubated overnight at 4 °C. After rinses with PBS, coverslips were incubated with appropriate Alexa-Fluor-488-conjugated and Alexa-Fluor-568-conjugated secondary antibodies for 1–2 h at room temperature, rinsed extensively in PBS, and mounted with ProLong Anti-fade media (Molecular Probes, Eugene, OR).

Sequential IF and smFISH were performed according to the manufacturer’s protocol (LGC Biosearch Technologies, Petaluma, CA) with the following modifications: IF was performed first. Cells were fixed for 10 min in 4% paraformaldehyde in 1x PBS, washed twice with 1x PBS, and permeabilized with 0.1% Triton X-100 in 1x PBS for 5 min at room temperature. Cells were washed once with 1x PBS and incubated with 70 µl of diluted primary antibody in 1x PBS for 1 hr at room temperature. Cells were washed three times with 1x PBS and incubated with 70 µl of diluted secondary antibody in 1x PBS for 1 hr at room temperature. Cells were washed three times with 1x PBS and post-fixed for 10 min in 3.7% formaldehyde in 1x PBS at room temperature. For the smFISH process, cells were washed with Wash Buffer A, incubated with 67 µl of Hybridization Buffer containing 125 nM DNA probes labeled with Quasar 670 (sequences in Supplementary file 1) for 6 hr at 37°C. Cells were then incubated with Wash Buffer A for 30 min at 37°C, Wash Buffer A containing 0.05 µg/ml DAPI for 30 min at 37°C, and Wash Buffer B for 3 min at room temperature. Coverslips were mounted using ProLong Antifade media (Life Technologies) and sealed with clear nail polish before imaging.

Coverslips were fixed for 30 min in 5% paraformaldehyde, washed and permeabilized with 0.1% TritonX-100 in PBS for 5 min. Coverslips were then incubated in 5% goat serum and Tuj-1 (R&D Systems, 1:200) or SMI-32 antibody (EMD Milipore, 1:500) in PBS overnight at 4°C. Coverslips were wash 3× and treated for 1 h with goat anti-mouse IgG conjugated to FITC or Rhodamine-Red (Invitrogen, 1:100) and 5% goat serum in PBS. Coverslips were mounted in Prolong antifade media (ThermoFisher Scientific) and viewed on a Nikon Eclipse inverted microscope for photography and cell counting.