4.6. Immunofluorescence Analysis

Adipose slices were subjected to microwave antigen retrieval in EDTA or citrate buffer and then gradually cooled. Samples were permeabilized with 0.5% Triton X-100 in PBS for 10 min and blocked with goat serum for 1 h at room temperature. The samples were incubated overnight at 4 °C with the antibody diluted in goat serum blocking solution. Anti-Collagen I (ab138492, Abcam, Cambridge, UK) and anti-Collagen VI (ab6588, Abcam) antibodies were used to identify the changes in ECM composition under normal conditions. Ninety-nine micrometer thick frozen sections of decellularized adipose tissue were blocked with goat serum in 0.1% Triton for 1 h at 37 °C. Then, samples were incubated overnight at 4 °C with Anti-Collagen I (ab138492, Abcam), Anti-Collagen VI (ab6588, Abcam), Anti-MMP9 (ab38898, Abcam) and Anti-MMP2 (ab92536, Abcam) antibodies diluted in goat serum. After three 30 min washes with PBS, samples were stained for 4 h at room temperature with fluorescent secondary antibodies (Abcam), followed by 10 min of DAPI staining for nucleus visualization. Slides were then viewed under a rotary laser confocal microscope (Revolution WD high-speed rotary laser confocal microscopy, Andor). Z-stack analysis was used to continuously image the decellularized ECM structure. Three-dimensional reconstruction was performed with the Imaris 9.0 software.