Immunohistochemical Analysis of Mouse Skin

Skin from mice was embedded in paraffin or snap frozen in OCT compound. Antigen retrieval for paraffin sections was performed in citrate buffer, pH6. Keratin 14 (MS-115; Neomarkers), Keratin 6 (PRB-169P; Covance), Keratin 10 (PRB-159P; Covance), TUNEL (Promega)/Active caspase-3 (9661; Cell Signalling) staining was performed on paraffin, and F4/80 (clone A3-1, homemade or MCA497G; Bio-Rad) staining on cryo sections. The staining was visualized with Alexa-488 or Alexa-567 conjugated secondary antibody. Measurement of epidermal thickness and inflamed area was done as described before (Jiao et al, 2020 (link)). Briefly, five optical fields per section were measured to quantify epidermal thickness. In each field, four measurements were performed. Percentage of inflamed area was determined as the percentage of inflamed versus total number of optical fields at 20× on individual skin section. Quantification of TUNEL and CC3 staining was performed on five optical fields per sections at 20× magnification. All images were acquired using either a Zeiss Meta 710 confocal, Leica SCN400 slide scanner or Leica DM5500 B microscope, and processed with ImageJ, Leica digital image hub or Leica Aperio ImageScope software.