Tsa plus fluorescence amplification kit

Immunostaining of tissue sections was performed as previously described [17 (link)]. IF was conducted using the antibodies p73 EP436Y (Abcam ab40658; 1:1000; Cambridge, UK) and p63α D2K8X (Cell Signaling Technology #13109; 1:1000; Danvers, MA), with TSA Plus Fluorescence Amplification Kit (Perkin Elmer, Waltham MA). Nuclei were counterstained with Slowfade Gold DAPI mounting media (Invitrogen, Carlsbad, CA). Micrographs of human tissues were all taken using a Zeiss slide scanner and further analyzed using Zeiss software. Micrographs of murine tissues were taken using a Leica microscope and camera and analyzed using Leica software; all scale bars = 50 µm.

Immunostaining of tissue sections was performed as previously described [3 (link)]. Murine skin tissues were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin for sectioning. De-identified human skin sections were obtained from pre-existing de-identified formalin-fixed paraffin-embedded tissue blocks. These blocks were prepared from excess tissue remaining after evaluation and diagnosis at the time of a surgical procedure. The Vanderbilt University Medical Center Institutional Review Board considers these tissues exempt since they were pre-existing and de-identified. IF was conducted using the following antibodies: p73 EP436Y (Abcam, ab40658, 1:1000), p63α D2K8X (Cell Signaling Technology, 13109, 1:1000), Keratin 5 (Fitzgerald Industries International, 20R-CP003, 1:200), Keratin 14 (Fitzgerald Industries International, 20R-CP002, 1:200), E-cadherin (BD Biosciences, 610181, 1:1000), Keratin 10 (Abcam, ab76318, 1:100), and Ki67 B56 (BD Biosciences, 550609, 1:1000). p73, p63, and Ki67 were detected using TSA Plus Fluorescence Amplification Kit (PerkinElmer). Keratins (5, 10, 14) and E-cadherin were detected using species-appropriate Alexa Fluor secondary antibodies at 1:200 (Thermo Fisher Scientific). IHC was conducted using γH2AX (Novus Biologicals, NB100-2280, 1:1000) antibody.