Immunofluorescence Staining of Tissue Sections

Tissue sections were first treated with 0.1 mg/ml protease and then incubated overnight at 4 °C with primary antibodies to anti-COL4 (C1926 Sigma) monoclonal antibody, anti-PDGFR-β, (1:200 dilution, AF385, R&D Systems), αSMA (1:500 dilution, Clone 1A4, Dako), glucose transporter-1 (GLUT-1, 1:200, Thermo Scientific). Sections were washed with PBS and further incubated with donkey anti-goat conjugated Alexa Fluor 594 (1:1000, A11058, Thermo Fisher Scientific, Waltham, MA, USA) and rabbit anti-mouse Alexa Fluor 488 (1:1000, A11059, Thermo Fisher Scientific). Sections were then washed in PBS before mounting in Vectashield with DAPI (H-1200, Vector Laboratories). Images were captured using a Leica TCS SP2 (upright) and Zeiss Spinning Disk (Invert) confocal microscopes as described previously [12 (link)].