Anti keratin 10

For K14, Ki67, K10 and Lef1 immunohistochemistry in human samples, paraffin sections were deparaffinized, rehydrated, followed by antigen unmasking performed for 20 min at 98 °C in citrate buffer (pH 6) using the PT module. Endogenous peroxidase was blocked using 3% H₂O₂ (Merck) in methanol for 10 min at room temperature. Endogenous avidin and biotin were blocked using the Endogenous Blocking kit (Invitrogen) for 20 min at room temperature. Nonspecific antigen blocking was performed using blocking buffer. Mouse anti-K14 (rabbit, 1/2000, Thermofisher), anti-Ki67 (rabbit, 1/400, Abcam, ab15580), anti-Keratin10 (rabbit, 1/200, Biolegend, ref.90541) and anti-Lef1 (rabbit, 1/100, Cell Signaling, ref.2230) were incubated overnight at 4 °C. Anti-rabbit biotinylated with blocking buffer, Standard ABC kit, and ImmPACT DAB (Vector Laboratories) was used for the detection of horseradish peroxidase (HRP) activity. Slides were then dehydrated and mounted using SafeMount (Labonord).

Skin tissues were embedded in paraffin or snap frozen in OCT compound. Antigen retrieval for paraffin sections was performed in citrate buffer, pH6 for the skin sections. Anti-F4/80 (clone A3-1, MCA497G, BIO-RAD), anti-Keratin 14 (MA5-11599, Invitrogen), anti-Keratin 6 (905701, Biolegend), anti-Keratin 10 (905401, Biolegend) were used for the staining. Alexa-488, Alexa-594 and Alexa-633 fluorescence conjugated secondary antibodies were used for detection. F4/80 staining was performed on cryo sections. All sections were counterstained with DAPI. Quantification of epidermal thickness was performed by measurement of epidermal thickness in five optical fields per section. 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 20x on individual skin sections. Assessment of tissue pathology was performed in a blinded fashion. All images were acquired using either a Zeiss Meta 710 confocal or PerkinElmer Spinning Disc confocal microscope for fluorescent images and brightfield images were acquired using a Leica SCN400 slide scanner or a Leica DM5500 B microscope.