Rnascope multiplex fluorescent detection reagents v2

Fluorescent in situ hybridization (FISH) was performed by using the RNAscope Multiplex Fluorescent Detection Reagents V2 (Advanced Cell Diagnostics, ACD, Hayward, CA, USA) according to the manufacturer’s instructions. RNAscope probe Mm-Hrh1 (491141) was purchased from ACD. Adult mice were perfused with ice-cold 4% PFA/PBS, and eyes were dissected out and fixed in 4% PFA/PBS at 4 °C overnight. The eyes were dehydrated with increasing concentrations of sucrose solution (10%, 20 and 30%) overnight before embedding in OCT on dry ice. Serial cross sections (12 µm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were pretreated with protease and then subjected to in situ hybridization with RNAscope Multiplex Fluorescent Detection Reagents V2 according to the manufacturer’s instruction (Advanced Cell Diagnostics, Hayward, CA). Briefly, sections were hybridized with the probe solution, followed by amplification and probe detection using TSA plus fluorophores (AKOYA, Marlborough, MA, USA). The sections were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were captured by a Zeiss LSM 880 confocal laser scanning microscope with 40×/1.0 Oil DIC (Carl Zeiss Microscopy, Thornwood, NY, USA).

Adult mice were perfused with ice-cold 4% PFA/PBS, and the eyeballs were dissected out and fixed in 4% PFA/PBS at 4 °C overnight. The eyeballs were dehydrated with increasing concentrations of sucrose solution (15–30%) overnight before embedding in optimal cutting temperature compound on dry ice. Serial cross sections (12 µm) were cut with a Leica cryostat and collected on Superfrost Plus Slides. The sections were pretreated with protease and then subjected to in situ hybridization with RNAscope Multiplex Fluorescent Detection Reagents V2 according to the manufacturer’s instruction (Advanced Cell Diagnostics, Hayward, CA, USA). Briefly, the sections were hybridized with the probe solution, followed by amplification and probe detection using TSA plus fluorescein/cyanine 5 (PerkinElmer, San Jose, CA, USA). The sections were mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Images were captured by Zeiss LSM 880 confocal laser scanning microscope with 63 × /1.40 Oil DIC (Carl Zeiss Microscopy, Thornwood, NY, USA). RNAscope probes Mm-Akt1, Mm-Akt2, and Mm-Akt1 were purchased from ACD and targeted bases 1130–2560, 2618–3665, and 24–1138 of mouse Akt1, Akt2, and Akt3 mRNA (NCBI reference sequence: NM_009652.3, NM_007434.4, and NM_011785.3), respectively.

Detection of Fkn/CX3CL1 mRNA using in situ hybridization was done to verify siRNA knockdown in phrenic motor neurons. On day 14, rats were anaesthetized under isoflurane anaesthesia (4% in O₂) and transcardially perfused with ice-cold RNase-free 1X phosphate buffered solution (PBS) followed by 4% paraformaldehyde. Spinal segments C3-C5 were harvested and dehydrated in RNase-free 30% sucrose. Twenty micrometer sections were cut on a microtome and collected in 10–15 sets of serial C3-C5 sections in RNase-free PBS, in order to obtain representative staining across segments C3-C5. Sections were mounted on to Superfrost Plus Gold microscope slides (ThermoFisher Scientific, Inc., Waltham, MA). After drying at room temperature for ~2 hours, slide-mounted tissue was stored at −80°C until use. All reagents used for in situ hybridization were purchased from Advanced Cell Diagnostics (Newark, CA). In situ hybridization was used for Fkn/CX3CL1 mRNA detection (reference no. 531141) using a fluorescence-based assay (reference no. 323110), as previously described^{89 (link)}. Experimental protocols were performed according to the manufacturer instructions in RNAscope Multiplex Fluorescent Detection Reagents V2 (Document No. UM 323100, Advanced Cell Diagnostics).