Alexa fluor 568 conjugated donkey anti rabbit antibody

For histological analysis, mouse ovaries were fixed in Bouin’s solution overnight, dehydrated in an ethanol gradient, embedded in paraffin, and cut into 5 μm sections. The sections were then deparaffinized, rehydrated, and stained with H&E following standard procedures. For IF experiments, WT and p63^+Δ/TID (or p63^+/R647C) ovaries or skin samples were fixed in 4% PFA, embedded in paraffin, and sectioned at 5 μm thickness. The slides underwent antigen retrieval and were then blocked, followed by incubation at 4°C overnight with the following primary antibodies: anti-DDX4 (1:600 dilution, Abcam, catalog ab27591); anti-p63 (1:200 dilution, Abcam, catalog ab124762); anti–cleaved-PARP1 (1:400 dilution, Cell Signaling Technology, catalog 94885S); anti-K10 (1:150 dilution, Abcam, catalog ab76318); anti-K14 (1:100 dilution, Santa Cruz Biotechnology, catalog sc-53253); and anti-Ki67 (1:200 dilution, Abcam, catalog ab15580). The slides were then washed in PBS, incubated with secondary antibodies (Alexa Fluor 568–conjugated donkey anti-rabbit antibody or Alexa Fluor 488–conjugated donkey anti-mouse antibody, 1:800 dilution, Invitrogen, Thermo Fisher Scientific, catalogs A21202 and A10042) for 1 hour at room temperature, and then sealed in antifade fluorescence mounting medium (Abcam). Images were captured with a fluorescence microscope (Olympus) and analyzed with ImageJ.