Methanol free formaldehyde

HBEpCs cells were grown on chamber slides overnight (Chamber slide ™, Lab-TekII, Thermo Fisher Scientific, Rochester, NY, USA) to 80–90% confluence. Cells were then exposed to IAV for 4 h. Subsequently, cells were washed with PBS and fixed with 4% methanol-free formaldehyde (Polysciences Inc., Warrington, PA, USA). Immunofluorescent staining was done as described earlier [23 (link)] and stained with an antibody that recognizes the phosphorylated form of NF-κBp65, rabbit anti-phospho-NF-κB p65 antibody (Millipore, Billerica, MA, USA) for 1 h, followed by Alexa-488 conjugated anti-rabbit secondary antibody (Life Technologies). HBEpCs overexpressing NFKBIB were infected with IAV and then stained with NFKBIB antibody (Cell signaling, Danvers, MA, USA) and NS1 antibody (Invitrogen, Foster City, CA, USA), followed by appropriate secondary antibodies (Alexa 488 and Alexa-555). The glass slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-Prolong Gold anti-fade reagent (Life Technologies) and protected with cover slips. Photomicrographs were made using a Zeiss Laser Scanning Microscopy (LSM)-510 (Carl Zeiss AG, Obertochen, Germany) confocal microscope.

Cell preparations on chamber slides were washed in pre‐warmed PBS, fixed in pre‐warmed 4% methanol‐free formaldehyde (Polysciences, Warrington, PA, USA), and washed in PBS. Samples were permeabilized in acetone at −20°C, washed, and treated with 5% normal goat serum in 1% (w/v) BSA in PBS to reduce nonspecific binding. Subsequently, cells were co‐incubated for 3 h at room temperature with rat anti‐ER‐TR7 (5 μg/mL; Abd Serotec) and rabbit anti‐COL3 (2 μg/mL; Abcam) or rabbit anti‐COL1 (1 μg/mL; Novus Biologicals). Primary antibodies were detected by indirect immunofluorescence using a goat antibody against the primary antibodies’ source species conjugated to Alexa dyes at 4 μg/mL with added DAPI at 300 mM. Samples were mounted under coverglass with Prolong Gold Antifade.