Rnascope ls fluorescent multiplex kit

RNA in situ hybridization experiments were performed using the RNAscope technology, which has been previously described (Wang et al., 2012 (link)). Paired double-Z oligonucleotide probes were designed against target RNA using custom software. The RNAscope LS Fluorescent Multiplex Kit and the probes targeting mouse pdgfr variant 1 and ly6a mRNA were from Advanced Cell Diagnostics (Newark CA). The RNAscope LS Fluorescent Multiplex Kit was used with standard pre-treatment conditions. FFPE mouse skeletal muscle tissue samples were incubated with Leica BOND Epitope Retrieval Solution 2 (ER2) at 95°C for 15 min. RNAscope 2.5 LS Protease III was used for 15 min at 40°C. Pre-treatment conditions were optimized for each sample and quality control for RNA integrity was completed using probes specific to the housekeeping genes Polr2a, Ppib, and Ubc. These probes are low, moderate, and high expressing genes, respectively. Negative control background staining was evaluated using a probe specific to the bacterial dapB gene. Fluorescent images were acquired using a 3D Histech Panoramic Scan Digital Slide Scanner microscope using a 40x objective.

RNA in situ hybridizations were performed by Advanced Cell Diagnostics, Newark, CA, using RNAscopeTM technology. Paired double-Z oligonucleotide probes were designed against target RNA using custom software. The probes used for rhesus macaque and mouse brain tissue samples are shown in table S11. RNAscope LS Fluorescent Multiplex Kit (Advanced Cell Diagnostics, Newark, CA, 322800) was used with custom pretreatment conditions following the instruction manual. Fixed frozen monkey and mouse fetal brain tissue slides were manually post-fixed in 10% neutral buffered formalin (NBF) at room temperature for 90 minutes. Then the slides were dehydrated in a series of ethanols and loaded onto the Leica Bond RX automated stainer, performing the reagent changes, starting with the pretreatments (protease), followed by the probe incubation, amplification steps, fluorophores, and DAPI counterstain. RNAscope 2.5 LS Protease III was used for 15 minutes at 40°C. Pretreatment conditions were optimized for each sample and quality control for RNA integrity was completed using probes specific to the housekeeping genes Polr2a, Ppib, and Ubc, which are low, moderate, and high expressing genes, respectively. Negative control background staining was evaluated using a probe specific to the bacterial dapB gene. Coverslipping was done manually using ProLong Gold mounting media at the end of each run.