Sab4501654

The different antibodies used were mouse monoclonal anti-K8 antibody (clone M20, Sigma-Aldrich, Darmstadt, Germany), mouse monoclonal anti-C terminal K8 antibody (clone 1E8, Abcam, Cambridge, UK), rabbit polyclonal anti-C terminal K8 antibody (SAB4501654, Sigma-Aldrich, Darmstadt, Germany), rabbit polyclonal anti-N terminal K8 antibody (ab137855, Abcam, Cambridge, UK), proprietary mouse monoclonal anti-K8 antibodies (clone D-A10 and clone D-D6), mouse monoclonal anti-uPA antibody (ab131433, Abcam, Cambridge, UK), mouse monoclonal anti-plasminogen antibody (ab38157 Abcam, Cambridge, UK), rabbit polyclonal anti-caspase 3 cleaved (Asp 175) antibody for IF analysis (9661, Cell Signaling Technology, Leiden, The Netherlands), rabbit monoclonal anti-cleaved caspase 3 antibody for IHC analysis (clone 5A1E, Cell Signaling Technology, Leiden, The Netherlands), mouse monoclonal anti-caspase 3 and cleaved caspase 3 Alexis antibody for Western blot (WB) analysis (Enzo Life Science, Farmingdale, NY, USA), mouse monoclonal anti-Ku80 antibody (ab3715, Abcam, Cambridge, UK), mouse monoclonal anti-histone H3 antibody (ab1791, Abcam, Cambridge, UK), anti-Na+/K+ ATPase alpha (H3) (SC-48345, Santa Cruz Biotechnology, Santa Cruz, CA, USA) rabbit polyclonal anti-GFP antibody (ab290, Abcam, Cambridge, UK), and mouse monoclonal anti-Ki67 antibody (Clone Mib1, M7240 DAKO, Santa Clara, CA, USA).

RNAscope in situ hybridization (ISH) was performed on decalcified paraffin tissue sections with RNAscope fluorescent multiplex reagent kit (ACD, 320850) according to the manufacturer’s protocol. Briefly, sections were baked at 60°C for 30 minutes, deparaffinized, treated with hydrogen peroxide, followed by antigen retrieval in target retrieval buffer for 15 minutes, and protease treatment before incubation with RNAscope probe for 2 hours at 40°C. After probe hybridization, RNA signal was amplified with Amp1, Amp2, and Amp3, followed by HRP-C3 incubation. Color development was performed with TSA plus Cyanine 3 kit (PerkinElmer, NEL744B001KT). The following RNAscope probe was used: nCoV2019-S (ACD, 848561-C3).
For dual ISH and IHC, immunofluorescence staining was carried out after RNAscope ISH according to the manufacturer’s protocol (ACD). Briefly, after ISH, sections were washed with PBS-T (0.1% Tween-20), blocked with 10% normal donkey serum at room temperature for 1 hour. Sections were then incubated with anti-NFH (chicken 1:5,000, Aves Labs, NFH) and anti-KRT8 (rabbit 1:500, Sigma-Aldrich, SAB4501654) overnight at 4°C, followed by incubation with secondary antibodies.