Sections from cryo-preserved human foreskin or 3D human skin equivalents and freshly isolated primary human keratinocytes were used. Antibodies detecting nuclear (active) phospho-NRF2 (S40) (Abcam, EP1809Y) or p63 (Abcam, clone 4A4) were applied overnight following 4% PFA fixation of the specimens. K10 and K14 antibodies were from DAKO, and the Ki-67 antibody (SolA15) was from eBioscience (Thermo Fisher Scientific). Fluorescently labeled secondary antibodies (Jackson ImmunoResearch) were used. Nuclei were counterstained with DAPI. Cells were counted using ImageJ (National Institutes of Health). p63- and pNRF2-stained sections were analyzed by confocal microscopy. Deconvolution of confocal images was performed using Leica software.
Clone 4a4
Manufactured by Abcam
Clone 4A4 is a monoclonal antibody. It is intended for research use only.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using clone 4a4
1
Immunohistochemical Analysis of Skin Samples
2
Immunohistochemical Analysis of Mammary Carcinomas
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The tumor samples were fixed in 10% formalin and embedded in paraffin, cut into 3 mm-thick sections, and then stained with hematoxylin-eosin-saffron (HES). Automated immunohistochemistry (Benchmark XT Ventana Medical Systems, Roche Diagnostics) was used on 3 mm-thick sections for confirmation of lymphovascular invasion using LMO2 as a lymphatic endothelium marker (LIM domain-only protein-2, clone SP51, Spring M351) and evaluation of invasiveness using p63 as a myoepithelial cell marker (clone 4A4, Abcam ab735). Mammary carcinomas limited by a continuous layer of p63-positive myoepithelial cells were defined as mammary carcinomas in situ and excluded from the present study. Mammary carcinomas with a focal interruption of their p63-positive myoepithelial cell lining, less than 1 mm long, were considered micro-invasive and also excluded from the present study. Mammary carcinomas lacking a p63-positive myoepithelial cell layer over at least 1 mm of their circumference were defined as invasive and were included. This technique was used only when invasiveness was doubtful on HES-stained slides.
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