Fluorescein isothiocyanate (fitc)

Cultured cardiac fibroblasts in 24-well-plate which was pretreated with poly-L-lysine were fixed for 30 min at room temprature in 4% paraformaldehyde, followed by three washes with phosphate buffered saline (PBS). Cells were permeabilized with 0.1% TritonX-100 in PBS for 10 minutes and blocked with 5% normal goat serum for 1 h. We used the following primary antibodies in our studies: vimentin (1:200 dilution, Sigma-Aldrich, Cat. No. V6389), α-actinin (1:1000 dilution, Sigma-Aldrich, Cat. No. A7811) and β₁-adrenergic receptor (1:200 dilution, Abcam, Cat. No. ab3442). After three washes with PBS, samples were probed with a fluorescently labeled TRITIC (1:200 dilution, ZSGB-BIO, Cat. No. ZF-0313) or FITC (1:200 dilution, ZSGB-BIO, Cat. No. ZF-0311) secondary antibody or for 1 h at room temperature. Following washout, cells were dyed using DAPI and imaged on the laser scanning confocal microscope for the cellular identification and expression of β₁-AR.

Cells were grown on sterile glass coverslips in 24-well plates treated as described in the figure legends. After 24 hours, cells were washed with cold PBS, fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% goat serum. Cells were then incubated with the primary antibody overnight at 4°C, and then with secondary antibody conjugated with FITC or TRITC (1:200; ZsBio). DAPI (Invitrogen) was used to visualize nuclei. The images were viewed using a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan).

Primary retinal pericytes were extracted from fetal bovine eyeballs and cultured in dulbecco's modified eagle medium (DMEM) containing 20% foetal bovine serum (FBS), as described previously [18 (link)]. All cells were cultured at 37°C in 5% CO₂, and the media were changed every 2–3 days. We use primary pericytes from passages 3–5 for this study. α-smooth muscle actin (α-SMA, Abcam, Cambridge, UK) and nerve/glial antigen 2 (NG2, Santa Cruz, Dallas, TX, USA) were used as positive stains to assess primary bovine retinal pericytes; factor VIII (Santa Cruz) and glial fibrillary acidic protein (Santa Cruz) were used as negative stains. The cells were immobilized with paraformaldehyde (4%) for half an hour, then infiltrated with Triton X-100 (0.5%) for 20 mins. The primary antibodies were incubated at 4°C overnight; the secondary antibodies combined with FITC (ZSGB-BIO, Beijing, China) were incubated at 37°C for 1 h. Finally, the cell nuclei were stained by 4′6-diamino-2-phenylindole (Beyotime, Shanghai, China), and the cells were detected by fluorescence microscopy (Life Technologies, EVOS FL Auto). Subsequently, the pericytes were pretreated with or without chloroquine (CQ, 10 μM, Sigma, St. Louis, MO, USA) for 2 h and then exposed to AGE-BSA (Biovision, San Francisco, CA, USA) or Control-BSA (Biovision) for 24 h.