Rabbit anti keratin 5

Frozen samples were sectioned at 6μm using with the cryostat (Leica Microsystems Inc, Buffalo Grove, IL). To remove endogenous mTomato signal, antigen retrieval was performed by heating the slides in Citra solution (BioGenex, Fremont, CA). The slides were blocked in 5% Normal Donkey Serum (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) in 5% PBST (phosphate buffered saline with 0.02% Tween-20) at room temperature for 1 hour, then stained with primary antibody diluted in blocking buffer overnight at 4°C. The next day, the slides were washed in PBS 3 x 10min, incubated in secondary antibody in blocking buffer for an hour at room temperature, and washed in PBS 3 x 10min before mounting on ProLong Gold DAPI (Life Technologies). The primary antibodies used were rabbit anti-Keratin 5 (1:1000, Abcam, Cambridge, MA), rat anti-Keratin 8 (1:250, Developmental Studies Hybridoma Bank, Iowa) and chicken anti- GFP (1:1000, Abcam). Anti-Rabbit-Cy3, anti-Chicken-AlexaFluor488, and Alexa Fluor 647-Strepravadin (all from Jackson ImmunoResearch) were used for the secondary antibody for immunofluorescent staining.

Section slides were fixed with 4% paraformaldehyde (Merck, Martillac, France), permeabilized with Tris-buffered saline (PBS) (Fisher Scientific, Illkirch-Graffenstaden, France) in the presence of 0.3% triton (Euromedex, Souffelweyersheim, France) and blocked for 1 h with PBS 5% BSA (Euromedex, France) plus 10% goat serum (Millipore, France). Sections were then incubated overnight at 4 °C with mouse anti-E-cadherin antibody (1/200, Becton Dickinson), rabbit anti-Keratin 1 (1/200, Abcam), rabbit anti-Keratin 10 (1/200, Abcam), rabbit anti-Keratin 5 (1/200, Abcam), and rabbit anti-Keratin 14 (1/200, Novus). After three PBS washes, the skin was incubated with secondary antibodies, Alexa 488 goat anti-mouse IgG (H + L) and Alexa 594 goat anti-rabbit IgG (H + L), respectively. After subsequent washing, the sections were mounted with Prolong Gold antifade reagent containing DAPI (Thermofisher Scientific, Illkirch-Graffenstaden, France), and images were acquired using Nikon confocal A1R (Nikon, Tokyo, Japan).

Cells were seeded on coverslips in 24‐well plates and fixed with 4% paraformaldehyde. After 15 mins, cells were permeabilized with phosphate‐buffered saline (PBS) containing 0.2% Triton X‐100 (PBST) for 5 min and then blocked with 1% bovine serum albumin in PBST. Immunostaining was performed using primary antibodies, rabbit anti‐cleaved‐Notch1 (Immunoway, plano, TX, USA) and mouse anti‐MCM5 (Proteintech, Chicago, IL, USA), rabbit anti‐Keratin 5 (Abcam, Cambridge, UK) and mouse anti‐Vimentin (Huaan Biotechnology, Hangzhou, China), respectively overnight at 4 °C. After PBS washing for three times, the slides were incubated with Alexa Fluor 555 donkey anti‐rabbit IgG and Alexa Fluor 488 goat anti‐mouse IgG (Beyotime, Beijing, China) at room temperature for 1 h. The slides were counterstained with DAPI (Beyotime, Beijing, China) and images were captured using the Zeiss microscope (Carl Zeiss, Jena, Germany).