Fitc conjugated collagen type 1

Primary human lung fibroblasts overexpressing flotillin-2–Myc or LacZ were fixed with cold 2% paraformaldehyde in PBS for 15 min. For localization of Endo180 and FLOT2, the internalized collagen study, primary human lung fibroblasts were incubated with FITC-conjugated collagen type I (25 μg/ml final concentration; Invitrogen) for 3 h at 37°C before fixation. Cells then were permeabilized with 0.1% Triton-X 100 in PBS (PBS-T) for 10 min, blocked with 5% normal goat serum in PBS-T, and incubated with primary antibodies to Myc (1:100) and Endo180 (1:50) or FLOT2 (1:100) and Endo180 (1:50) overnight at 4°C, followed by Alexa Fluor 488– and Alexa Fluor 594– or Alexa Fluor 647– and Alexa Fluor 594–conjugated secondary antibodies (1:100; Invitrogen) for 1 h at room temperature. Confocal images were captured by a Leica point scanner using a Leica TCS SPE confocal microscope with an ACS APO 63× Oil CS objective lens at room temperature with Leica Type F Immersion Liquid (11513859). Images were processed with Photoshop CS6 software (Adobe, San Jose, CA).