Alexa fluor 594 conjugated anti rabbit

Immunofluorescence was used to detect the expression of collagen II in each group transfected with and without the FGF-2 gene. P3 hAMSCs were seeded onto sterile cover slips of 6-well plates at a density of 10⁵ cells/ml (3 wells per group), followed by addition of lentivirus at the optimal MOI. Fourteen days later, after observing intense fluorescence under an inverted fluorescence microscope, the hAMSCs were fixed with 4% paraformaldehyde for 15 min at room temperature. Wells were blocked with 5% goat serum for 60 mins, and the hAMSCs were incubated overnight at 4°C with the primary antibodies against collagen type II (1 : 200, ab34712, Abcam). The next day, hAMSCs were incubated with Alexa Fluor® 594 conjugated anti-rabbit (Abcam, USA) secondary antibodies for 1 hour at room temperature, and cell nuclei were stained using 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI). The results were observed using an inverted fluorescence microscope. Fourteen days after transfection of the FGF-2 gene, hAMSCs of each group were fixed with 4% paraformaldehyde for 15 mins at room temperature followed by washing thrice with PBS. TB (Sigma, USA) staining was performed, and results were observed under an inverted microscope.

After treatment, cardiofibroblasts were washed with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Then cells were blocked by 5% donkey serum (Sigma-Aldrich, St Louis, MO) for 1 h at room temperature after rinsing thoroughly. Cardiofibroblasts were incubated overnight at 4°C with anti-S100A4 (1:100; Proteintech, Wuhan, China) and anti-α-SMA (1:200; Abcam). Sections were then incubated with fluorescent secondary antibodies conjugated with Alexa Fluor 594-conjugated anti-rabbit (1:400; Abcam) and Alexa Fluor 488 anti-mouse (1:400; Abcam) for 1 h at room temperature. Nuclei were counterstained with 4’,6 diamino-2-phenylindole (DAPI, Abcam). Images were captured though laser scanning confocal microscopy (ECLIPSE-Ni, Nikon, Japan).

Tissues were fixed in 4% paraformaldehyde (PFA) for 24 h, and then dehydrated and embedded in paraffin. The paraffin blockers were cut into 5 μm sections and stained with hematoxylin and eosin. The H&E images were taken from histological light microscopy (Olympus BX51) and adipocyte diameter was measured by imageJ software.
For immunofluorescence staining, SVF-derived beige adipocytes were washed twice with PBS and then fixed with 4% paraformaldehyde (PFA) for 20 min at 22 °C. After washing, the fixed cells were permeabilized with 0.5% Triton X-100 (Lablead) for 20 min and then blocked with 5% BSA for 30 min at 22 °C. After washing, the adipocytes were incubated with anti-PRDM16 (R&D, 1:50), anti-YAP (CST, 14074, 1:100) or anti-TAZ (CST, 83669, 1:100) at 4 °C overnight. After washing with PSBT, the adipocytes were incubated with Alexa Fluor 488-conjugated anti-sheep (Invitrogen, A11015, 1:300) and Alexa Fluor 594-conjugated anti-rabbit (Abcam, ab150080, 1:300) for 1 h at 22 °C, followed by counterstaining with DAPI (CST, 1:5000). Adipocytes were imaged using Nikon A1RSi+ Confocal Imaging Systems for Fluorescence.