Uv meta 510 confocal microscope

Fibroblasts were pretreated with roscovitine (5 μM) for 15 min, followed by incubation in media with 10 ng/ml TGF-β2. Twenty-four h later, cells were fixed in 4% paraformaldehyde, washed in phosphate buffered saline (PBS), and incubated with primary antibodies against αSMA (1: 400 dilution, Abcam) or Type I collagen (1:200, Southern Biotechnology) for 120 min, followed by Alexa^® 594-conjugated secondary antibodies (Invitrogen) for 60 min. Nuclei were identified by 4›-6-diamidino-2-phenylindole (DAPI) staining. Non-immune IgG was used as a negative control in each experiment. Following stringent washing, slides were examined under a Zeiss UV Meta 510 confocal microscope (Carl Zeiss, Jena, Germany). Each experiment was repeated at least three times with consistent results.

Fibroblasts seeded on 8-well Lab-Tek II chamber glass slides (Nalgene Nunc International, Naperville, IL, USA) were incubated in serum-free DMEM with 0.1% BSA for 24 h. Fresh media with AdipoRon were added for 24 h. Cells were fixed, permeabilized, and incubated with primary antibodies to α-SMA (Sigma-Aldrich) and type I collagen, both at 1:500 followed by Alexa-fluor-labelled secondary antibodies (Invitrogen, Carlsbad, CA, USA). Nuclei were identified using 4,6-diamidino-2-phenylindone (DAPI) in ProLong Gold antifade reagent (Invitrogen). Immunofluorescence was evaluated under a Zeiss UV Meta 510 confocal microscope (Carl Zeiss Inc, Jena, Germany).