Paraffin-embedded histological sections were stained with H&E for determination of the histological changes in epidermal and tongue epithelium in Grhl2 WT and KO mice, and were analyzed without blinding by an oral pathologist. Mouse oral mucosa and skin were fixed in 4% (wt/vol) paraformaldehyde at 4 °C for 24 h. Samples were embedded in paraffin, sectioned at 4 μm thickness, and stained as described previously7 (link). Numbers of positive staining cells were counted and plotted as % of all cells in at least 10 fields in each slide.
For IFS, cells were cultured in Nunc™ Lab-Tek™ II Chamber Slide™ System (ThermoFisher Scientific) to reach 70–80% confluence and fixed in 2% paraformaldehyde for 20 min. Cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min, and then blocked for 1 h in PBS containing 2% FBS, and incubated overnight at 4 °C with the primary antibody. After three washes with PBS, cells were incubated with the secondary antibody for 1 h. Slides were mounted in Prolong Gold w/DAPI (Invitrogen). Images were captured on an Olympus epifluorescence inverted microscope (Olympus, Cypress, CA). IFS signals were quantified via Image J software and the corrected total cell fluorescence was calculated.
For IFS, cells were cultured in Nunc™ Lab-Tek™ II Chamber Slide™ System (ThermoFisher Scientific) to reach 70–80% confluence and fixed in 2% paraformaldehyde for 20 min. Cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min, and then blocked for 1 h in PBS containing 2% FBS, and incubated overnight at 4 °C with the primary antibody. After three washes with PBS, cells were incubated with the secondary antibody for 1 h. Slides were mounted in Prolong Gold w/DAPI (Invitrogen). Images were captured on an Olympus epifluorescence inverted microscope (Olympus, Cypress, CA). IFS signals were quantified via Image J software and the corrected total cell fluorescence was calculated.