Dapi fluromount g

Harvested tissue was immediately embedded in OCT, sectioned at 10 µm, and mounted onto glass slides. Slides were rinsed in phosphate buffered saline (PBS, Gibco, 10010023), incubated with 10 µM of dihydroethidium (DHE, ThermoFisher Scientific D1168) in a light-protected humidified incubator at 37˚C for 30 min, rinsed in PBS, and mounted with slide coverslips using DAPI Fluromount-G (SouthernBiotech, 0100-20). Images were taken using the LSM 880 inverted confocal microscope (Airyscan, GaAsP detector, 880, Beckman) immediately. Staining was quantified by measuring the average intensity of ten randomly selected images per group on ImageJ.

Immunohistochemistry was performed on 8 μm sectioned paraffin slides which were blocked with 1X Powerblock (HK083‐50 K; Biogenex, Fremont, CA) and incubated for 1 h at 37°C with the relevant primary antibody for 1 h at 37°C. Specimens were washed in phosphate‐buffered saline (10,010,023; Gibco®, Dublin, Ireland) and incubated with a secondary antibody for 1 h at 37°C. Specimens were then washed in phosphate‐buffered saline (10,010,023; Gibco®, Dublin, Ireland) and mounted onto glass slides in DAPI Fluromount‐G (0100–01; SouthernBiotech, Birmingham, AL). Fluorescent images of the dermal layer were taken using an SP8 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) at 20X magnification (25 images per condition). The percentage of stain‐positive pixels was calculated on each image using ImageJ with a Colour Detect macro to quantify the relevant number of pixels.

Following dissection, specimens were washed in phosphate buffered saline (PBS) three times and mounted on onto glass slides with DAPI Fluromount-G (0100-20; SouthernBiotech, Birmingham, AL). Confocal microscopy imaging was performed with a LSM 880 inverted confocal microscope (Leica Microsystems, Wetzlar, Germany) with the 20X objective) located in the Cell Sciences Imaging Facility (Stanford University, Stanford, CA). Raw image stacks were into imported into ImageJ (National Institutes of Health, Maryland, USA) for analysis.