Rabbit anti ki 67

For intracellular staining of ALB and AAT, the cells were fixed with 4 % paraformaldehyde for 15 minutes at room temperature and then incubated with PBS containing 0.2 % Triton X-100 (Sigma) for 15 minutes. Cells were then washed three times with PBS. After being blocked by 3 % bovine serum albumin (BSA) in PBS for 60 minutes at room temperature, cells were incubated with primary antibodies at 4 °C overnight, washed three times with PBS, and then incubated with appropriate fluorescence-conjugated secondary antibody for 60 minutes at 37 °C in the dark. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma). Primary and secondary antibodies were diluted in PBS containing 3 % BSA. Antibodies used for immunofluorescence are as follows: sheep anti-ALB (1:500; Bethyl), rabbit anti-AAT (1:1; Abcam, Chengdu, Sichuan, P.R. China), rabbit anti-hepatocyte nuclear factor alpha-4 (anti-HNF4α, 1:50; Santa Cruz), rabbit anti-Ki67 (1:100; Santa Cruz), dylight 488 conjugated donkey anti-rabbit IgG (1:200; Bethyl), dylight 594 conjugated donkey anti-sheep IgG (1:200; Bethyl), and Alexa Fluor 594 conjugated goat anti-rabbit IgG (1:100; ZSBG-BIO, Chengdu, Sichuan, P.R. China).

Analysis included tissues obtained from three to five mice per genotype from two or more litters. For immunofluorescent staining, tissues were dehydrated and then stained as detailed previously (Amitai-Lange et al., 2015 (link)). Primary antibodies were rabbit anti-K10 1:400 (Covance, PRB-159P), mouse anti-K14 1:200 (Millipore, CBL197), mouse anti-FLG 1:75 (Abcam, AB31356), mouse anti-P63 1:100 (Santa Cruz, sc-8431), rabbit anti-Ki67 1:100 (Santa Cruz, sc-7846), mouse anti-K15 1:200 (Santa Cruz, sc-47697), and goat anti-FIH1 1:100 (Santa Cruz, sc-26219). In situ hybridization was performed as described previously (Shalom-Feuerstein et al., 2012 (link)). To quantify the percentage of Ki67-positive cells among DAPI-positive epidermal basal layer cells, a total of 500 cells of 5 different regions on 5 different slides were manually counted and quantified using ImageJ. Epidermal thickness was measured by using the line selection tool in ImageJ 1.48v using 10 different skin regions in each slide and a total of 30 regions were measured. Similarly, to determine the amount of K14- and K10-positive cells for each genotype, nuclei of DAPI and K10 or K14 co-stained cells were counted in epidermal fields that were defined for the length of the dashed lines indicating the dermal-epidermal border.