Mtor monoclonal antibody

Cells from the co-culture and homogenized mouse ear tissue were lysed with RIPA lysis buffer (Thermo Fisher Scientific). Protein content from cell lysis and mouse ear tissue was determined with protease inhibitor and phosphatase inhibitor (Sigma-Aldrich) and Pierce BCA protein assay (Thermo Fisher Scientific). AMPK alpha-1 monoclonal antibody, mTOR monoclonal antibody and IL-37 polyclonal antibody were obtained from Thermo Fisher Scientific. SQSTM1/P62, β-actin rabbit mAb, HRP-conjugated anti-rabbit IgG and HRP-conjugated anti-mouse IgG were obtained from Cell Signaling Technology. Dorsomorphin (Compound C/CC AMPK inhibitor) and LC3B were obtained from Sigma-Aldrich Corp. Secondary antibodies were used at 1:2000 and other primary antibodies were diluted at 1:1000.

Sections (5 mm) were stained with H&E to assess the general morphology. Paraffin sections were immunostained with lineage-specific antibodies to identify eosinophils and dermal fibroblasts by immunohistofluorescence. Samples were incubated with rat anti-mouse major basic protein (MBP) antibody (specific for eosinophils, a gift from James J. Lee, Ph.D., Mayo Clinic, Scottsdale, AZ, United States), rabbit anti-Vimentin antibody (specific for dermal fibroblasts, Cell Signaling Technology, Beverly, MA, United States), AMPK alpha-1 monoclonal antibody, mTOR monoclonal antibody, and IL-37 polyclonal antibody (Thermo Fisher Scientific, Rockford, IL, United States), and LC3B antibody (Sigma-Aldrich, St. Louis, MO, United States). Cy3-conjugated goat anti-rat immunoglobulin G (IgG) antibody (Beyotime Co., Shanghai, China) and Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse IgG antibody (ABclonal, MA, United States) were used as secondary antibodies. All images were acquired with a Leica DM6000B microscope (Leica Microsystems GmbH, Wetzlar, Germany) and processed using the Leica Application Suite software (Leica Microsystems GmbH).