Sc 23877

Keratinocytes were grown on glass coverslips, fixed with 3,8% formaldehyde in PBS for 10 minutes and permeabilized with cold methanol for 5 minutes or fixed with cold methanol for 10 minutes. Paraffin-embedded skin sections were prepared following convential procedures. Samples were incubated for 1 hour (cells) or overnight (sections) in a wet chamber with antibodies against keratin 10 (sc-23877), phospho-histone H3 (sc-8656-R), cyclin A (sc-56299), cyclin E (sc-198), αTubulin (SC-23948) (all from Santa Cruz, CA, USA), involucrin (I-9018, Sigma-Aldrich, Inc), keratin 1 (PRB-149P, Covance, Vienna, VA, USA) or Ki67 (ab16667, Abcam, Cambridge, UK). Then, primary antibodies were revealed with Alexa Fluor-conjugated goat anti rabbit or anti mouse antibodies (Jackson ImmunoResearch, PA, USA). Samples were then incubated with 0.2 µg/ml DAPI (Vector Lab, Burlingame, CA). Coverslips were mounted with Prolong Gold Antifade Reagent (ThermoFisher Scientific, Waltham, MA) and cells were visualized and photographed with a Zeiss fluorescent microscopy. Immunofluorescence staining was scored by counting 400 cells.

The following antibodies were used: anti-Cdk1 (610038, BD Biosciences; immunohistochemistry (IHC)), anti-filaggrin (PRB-417P, Covance; immunofluorescence (IF)), anti-involucrin (RINVOL, 924401 Biolegend; FC and IF; SY3 [65 (link)], FC), anti-IgG (I-5381, Sigma-Aldrich; FC), anti-K1 (PRB-149P, Covance; FC), anti-K5 (SAB45016501, Sigma-Aldrich; IF), anti-K10 (C7284, Sigma-Aldrich; FC, sc-23877, Santa Cruz Biotechnology; IF and PRB-159P, Covance; IF), anti-K13 (sc-390982, Santa Cruz Biotechnology; IF), anti-K16 (LL025, Santa Cruz Biotechnolog; FC), anti-Ki67 (sc-7844, Santa Cruz Biotechnology; IF), anti-Plk1 (laboratory made; [18 (link), 23 (link)] IHC), anti-phospho-histone H3 Ser10 (9701, Cell Signalling; IHC) and anti-PCNA (sc-56, Santa Cruz Biotechnology; IF).
The following secondary antibodies were used: Alexa Fluor® 488-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Jackson; FC and IF), Alexa Fluor® 594-conjugated goat anti-rabbit or anti-mouse IgG antibodies (Jackson; IF).