Immunofluorescence staining.

Immunofluorescence microscopy was performed as described previously (32). Briefly, 5-μm tissue sections on charged glass slides (VWR no. 48311-703) were baked for 2 h at 60°C and cooled to room temperature for 30 min prior to staining. Deparaffinized tissue sections in xylene were hydrated with a decreasing ethanol gradient (100% to 50%). Antigen retrieval in 10 mM sodium citrate buffer, pH 6.0, and 0.05% Tween 20 was performed using a pressure cooker for 9 min. Slides were permeabilized with 0.5% Triton X-100 in 1× PBS for 45 min at room temperature and incubated with blocking buffer (5% bovine serum albumin [BSA] in 1× PBS) for 1 h at room temperature in a humidified chamber. Primary antibody was added to 2.5% BSA in 1× PBS and incubated overnight at 4°C in a humidified chamber. Primary antibodies used include antibodies specific for pRb (1:25, BD Pharmingen no. 554136), involucrin (1:100, Santa Cruz no. sc-28557), K10 (1:50, Santa Cruz no. sc-52318), KLF4 (1:100, Sigma no. HPA002926), PRDM1 (1:50, Cell Signaling no. 9115), PCNA (1:300, Santa Cruz no. sc-56), cyclin B1 (1:100, Abcam no. 32053), p107 (1:50, Santa Cruz no. sc-250), and p130 (1:100, Sigma-Aldrich no. HPA019703). Tissue sections were extensively washed and incubated with secondary antibodies (Invitrogen no. A11029, A11032, A11034, and A11058) diluted 1:1,000 in 1× PBS for 1 h at 37°C in a humidified chamber. For sequential staining, slides were incubated with a poly-horseradish peroxidase (poly-HRP)-conjugated antibody (Invitrogen no. {"type":"entrez-nucleotide","attrs":{"text":"B40961","term_id":"2545213","term_text":"B40961"}}B40961) for 1 h at 37°C after the first primary antibody incubation. Tissues were then incubated with Opal dyes (Akoya no. FP1495001KT) or tyramide-488 (Invitrogen no. {"type":"entrez-nucleotide","attrs":{"text":"B40953","term_id":"2545205","term_text":"B40953"}}B40953) for 10 min at room temperature and protected from light. Antigen retrieval was then performed using a pressure cooker for 15 min with the buffer previously outlined. After cooling for 20 min, the slides were incubated with blocking buffer and a second primary antibody as previously described. Following the second primary antibody incubation, tissues were incubated with the poly-HRP antibody and Opal dyes as mentioned above. Tissues were mounted with ProLong diamond antifade mountant with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen no. {"type":"entrez-protein","attrs":{"text":"P36962","term_id":"547832","term_text":"P36962"}}P36962). For slides stained with Hoechst, a 1:1,000 dilution of Hoechst (Thermo Fisher no. 62249) was added at the time of secondary antibody or Opal dye incubation and mounted with Invitrogen ProLong glass antifade mountant (no. {"type":"entrez-protein","attrs":{"text":"P36980","term_id":"543983","term_text":"P36980"}}P36980). Slides were imaged on a Zeiss Axio Observer Z1 inverted fluorescence microscope in the Research Core Microscopy Imaging Core at LSU Health Shreveport. Images were equally brightened with ImageJ. For quantification, images were manually quantified or quantified with ImageJ when indicated.