Viewrna ish tissue assay kit

The hearts of male C57/B6 mice were dissected, immersed immediately in fresh 10% neutral buffered formalin, fixed for 16 hours at room temperature, rinsed briefly, dehydrated and embedded in paraffin. Heart were cut into 6 μm transverse sections. The TYPE-1 ViewRNA probe against Chaer LncRNA was designed by Affymetrix and was detected by ViewRNA™ ISH Tissue Assay Kit (Affymetrix, QVT0012) according to manufacturer's instructions. Briefly, tissue sections were pretreated in 90-95 °C pretreatment solution for 10 minutes, followed by protease incubation at 40°C for 35 minutes. Hybridization was performed at 40°C for 2-4 hours. After the preamplifier, amplifier and label probe 1-AP hybridization steps, probes were visualized by fast red substrate at 40°C for 40 minutes. Immunofluorescence assays were performed subsequently to detect the cardiac myocytes, endothelial cells and fibroblasts by antibodies against Troponin T Type 2 (Tnnt2; Bioworld Technology, BS6013, 1:100), isolectin b4 (ib4; Enzo Life Sciences, ALX-650-001B-MC05, 1:100) and type I collagen (MD Biosciences, 203002, 1:200), respectively. Images were taken by Olympus FluoView™ FV1000 confocal laser scanning microscope Brochure.

Formalin-fixed paraffin-embedded tissue was sectioned (4μm) and analyzed for SERPINB5 and Cytokeratin-19 (KRT19) expression using ViewRNA™ ISH Tissue Assay Kit (ThermoFisher), with probes against SERPINB5 (VA1–12247-VT) and cytokeratin-19 (VA6–10947-VT) (ThermoFisher), according to manufacturer’s protocol. In short: tissue was deparaffinized in Xylene and 100% ethanol. The sections were pretreated for 10min at 90–95°C, followed by protease digestion for 10min at 40°C and then fixed in 10% normal buffered formalin followed by hybridization with the target probes against SERPINB5 and KRT19 for 2h in 40°C. The sections were stored overnight in storage buffer before proceeding with signal amplification and detection. The sections were preamplified for 25min at 40°C followed by amplifier hybridization for 15min at 40°C. Followed by incubation with label probes at 40°C for 15min and addition of substrate. FastRed label probe and substrate incubation was performed first. Sections were then counterstained in Gil’s hematoxylin, dipped in 0.01% ammonium hydroxide followed by DAPI staining (1μg/ml for 10min). Slides were mounted with Prolong gold antifade mountant with DAPI (Thermofisher). The confocal images were taken on a Zeiss LSM 710 microscope and the IHC images were scanned on the 3DHISTECH Pannoramic 250 flash III.