Ms 113 p

Tissue sections (skin and fat) were stained with markers for macrophages (CD68, DAKO/Agilent M0814, CA, USA), mast cells (CD117, BioCare CM5296C, CA, USA), T lymphocytes (CD3, Leica NCL-L-CD3-565, IL, USA), endothelial cells (CD31, DAKO/Agilent M0823, CA, USA), blood vessels (SMA, Thermo MS-113-P, MA, USA), and lymphatic vessels (D2-40, Biolegend SIG-3730, CA, USA), counterstained with hematoxylin; positive and negative controls were included (San Diego Pathologists Medical Group, CA, USA). Angiogenesis was assessed by Masson's trichrome stain (Sigma kit HT102A-1KT, MO, USA). All sections were coded and scored in a blinded manner by two independent investigators. The quantification of CD3, CD68, and CD117 positive cells was expressed as the average of total number of cells counted in 10 fields at 20x magnification (five fields in skin and five in fat).

Kidneys were embedded in OCT (4583, SAKURATissue-Tek®, Torrance, CA, USA) on dry ice. Five-micrometer sections were cut and performed with immunostaining. Briefly, the slices were fixed in 10% neutral buffered formalin; then washed with PBS and treated with 0.2% Triton X-100; mouse anti-αSMA (MS-113-P, Thermofisher, USA) and rabbit anti-synapotodin (sc-50459; Santa Cruz Biotechnology, Santa Cruz, CA, USA); or mouse anti-collagen 1α (ab6308, abcam, USA) and rabbit anti-synapotodin; or mouse anti-fibronectin (sc-271098, Santa Cruz, USA) and rabbit anti-synapotodin; or mouse anti-TGFβ (MAB1835, R&D, USA) and rabbit anti-synapotodin were incubated with the slices after blocked with 1% BSA; Donkey anti-mouse IgG-488 (ab150105; Abcam, Shanghai, China) and donkey anti-rabbit IgG-647 (ab150075; Abcam) were incubated with the slices; Hoechst 33342 was then used to stain the nucleus; the slices were mounted in VECTASHIELD Mounting Medium (H-1000, Vector Laboratories, Inc., Burlingame, CA, USA) staining. Images were obtained using a confocal microscope (TCS-SP8; Leica, Buffalo Grove, IL, USA). Positive cells with both synapotodin and EMT markers located in the glomeruli were counted and divided by synapotodin-positive cells located in the glomeruli. At least 100 glomeruli were analyzed in each group.