Probe hybridization and subsequent immunofluorescent staining

Probe hybridization closely followed the manufacturer’s instructions (Advanced Cell Diagnostics). Sections were immersed in pre-chilled 4% PFA for 15 min at 4 °C. They were then dehydrated at room temperature (RT) in 50%, 70% and 100% ethanol (2X) for 5 min each and allowed to dry for 1–2 min. Fixation and dehydration was followed by protease digestion, using Protease 4 for 30 min at RT. Sections were then incubated at 40 °C with the following solutions: 1) target probe in hybridization buffer A for 3 hours; 2) preamplifier in hybridization buffer B for 30 minutes; 3) amplifier in hybridization buffer B at 40 °C for 15 minutes; and 4) label probe in hybridization buffer C for 15 minutes. After each hybridization step, slides were washed with wash buffer three times at RT. For fluorescent detection, the label probe was conjugated to Alexa Fluor 488. Probes for K⁺ channels and a blank negative control were obtained from Advanced Cell Diagnostics. Sequences of the target probes (for the specified K⁺ channels), preamplifier, amplifier, and label probe are proprietary. Detailed information about the probe sequences can be obtained by signing a nondisclosure agreement provided by the manufacturer.

For subsequent immunofluorescent staining, slides were treated with 10% blocking solution for 10 min at RT, incubated with anti-Tubulin β3 (TUJ1, BioLegend, 1:300 dilution), overnight at 4 °C, washed with PBS three times for 5 min each, incubated with the appropriate Alexa Fluor secondary antibody (ThermoFisher) diluted 1:500 for 2 hours at RT, and again washed with PBS three times for 5 min each. Incubation in Hoechst 33342 solution for 15 s at RT was performed to label cell nuclei. Slides were then mounted in Fluoromount-G and sealed under a coverslip.

Confocal micrographs were obtained as described below. Individually fluorescently labelled mRNA transcripts appeared as puncta. To quantify the number of mRNA transcripts per SGN, individually fluorescently labelled mRNAs within a given field of view (FOV) were detected using the spots function in Imaris 6.4 software (Bitplane). mRNA counts were normalized to the number of TUJ1-labeled SGNs marked manually in the same FOV.