Procartaplex universal assay buffer

Skin samples were snap-frozen in liquid nitrogen immediately after sacrifice and stored at −80°C. To prepare whole skin lysate, frozen samples were diced using a scalpel then 500ul of lysis buffer (RIPA with 1mM PMSF) was added per 100mg of sample and two sterile 4.5mm ball-bearing were added to the tube prior to mechanical dissociation using the TissueLyserII (Qiagen): 25 beats/s for 3 minutes, repeated up to 3 times until homogenized. Lysate was centrifuged at 4C for 10 minutes at 16,000xg to remove unhomogenized debris and then supernatant was aliquoted and frozen at −80°C. Total protein was measured using a Bradford Assay (BioRad). Skin lysates were diluted 1:10 in ProcartaPlex Universal Assay Buffer prior to use in custom Procartaplex Immunoassay (ThermoFisher) analyzed using the FlexMap3D (Luminex). Skin lysates were subjected to acid-ethanol extraction (Flanders et al. 2016 (link)), lyophilized and resuspended in ProcartaPlex Universal Assay Buffer and stored at −80°C prior to measurement of TGF-B using Procartaplex Single-plex assay (ThermoFisher).