Rnascope multiplex fluorescent detection kit

Fluorescence in situ hybridization was performed with probes targeting Prrx1 (GenBank accession #NM_011127.2) mRNA by using the RNAscope Fluorescent Multiplex Detection Kit (Advanced Cell Diagnostics) according to the manufacturer instructions and as described previously (Wang et al., 2018 (link), Saraswat et al., 2021a (link)). Fixed sections were baked at 60°C for 30 minutes, washed with ethanol, tissue pretreatment with proprietary buffers. Probes were then hybridized and fluorescent conjugated. Sections were counterstained with DAPI to visualize nuclei. Positive signals were identified as punctate dots present in nucleus and cytoplasm.

Control and YTHDC1 depleted cells were fixed with 4% paraformaldehyde and permeabilized with cold methanol. Fixed cells were then cytospun onto glass slides. RNA in situ hybridization was performed using RNAscope multiplex fluorescent detection kit according to the manufacturer’s instructions (Advanced Cell Diagnostics). RNAscope probes targeting human MYC was designed and produced by ACDbio. After the in stiu hybridization was completed, slides were washed twice and subjected to immunostaining.
For immunostaining, cells were fixed with 4% paraformaldehyde 15 mins at room temperature and permeabilized with cold methanol. Fixed cells were then cytospun onto glass slides and were blocked (with buffer PBST+0.5%BSA) for 1h followed with staining on slides with primary antibody and secondary Ab (Goat anti–rabbit Alexa flour 488, Invitrogen) with DAPI counterstaining. Antibodies used are shown in the resource table.

For mouse Muc2 and Ramp1 RNAscope staining, ileums and colons were collected, embedded in OCT and frozen. 14μm sections were fixed with 4% PFA at 4 °C for 15 min and stained for Muc2 and Ramp1 using the RNAscope Multiplex Fluorescent Detection Kit (Advanced Cell Diagnostics) following the manufacturer’s instructions. Entire sections were imaged using a confocal microscopy at 20X and images were analyzed in FIJI.
For human MUC2 and RAMP1 RNAscope staining, 5 μm paraffin-embedded sections were deparaffinized, treated with hydrogen peroxide for 10 minutes, and boiled in RNAscope Target retrieval buffer for 15 minutes. The sections were then stained for MUC2 and RAMP1 using RNAscope Multiplex Fluorescent Assay v2 (Advanced Cell Diagnostics) following the manufacturer’s instructions. Entire sections were imaged using a confocal microscopy at 20X and images were analyzed in FIJI.