Mouse anti keratin14

Skin and buccal mucosa specimens were frozen in an optimal cutting temperature (OCT) compound (Cryomount I, 33351; Muto Pure Chemicals, Japan) and sectioned with a cryostat (Leica CM1950, Leica Biosystems, Germany). To prepare horizontal‐sliced sections, specimens were embedded horizontally in the OCT compound, and 5 μm‐thick sections were prepared. Frozen sections were incubated with primary antibodies: rabbit anti‐ZO‐1 (dilution 1:200 in PBS, ab2168; Abcam, UK), rabbit anti‐claudin‐1 (dilution 1:200 in PBS, ab15098; Abcam, UK), rabbit anti‐occludin (dilution 1:100 in PBS, 71‐1500; Invitrogen, CA), mouse anti‐keratin14 (dilution 1:200 in PBS, ab7800; Abcam, UK), rabbit anti‐keratin10 (dilution 1:500 in PBS, 905404; BioLegend, CA), rabbit anti‐cleaved caspase‐3 (dilution 1:100 in PBS, #9661 S, Cell Signalling Technology, MA) or anti‐Ki‐67 (dilution 1: 200, NB600‐152, Novus Biologicals) at 37°C for 60 min. The sections were subsequently incubated with Fluorescein isothiocyanate (FITC)‐conjugated goat anti‐rabbit IgG H + L (dilution 1:200 in PBS, 234; MBL), alexa 488‐conjugated anti‐mouse IgG3 (dilution 1:1000 in PBS) or alexa 568‐conjugated anti‐rabbit IgG (dillution 1:1000 in PBS) at 37°C for 60 min and mounted in a Pharma Fluor aqueous mounting medium (TA‐006‐FM; Thermo Fisher Scientific, MA). The stained immunofluorescent samples were observed with a BZ9000 microscope.