Anti αsma mouse monoclonal antibody

Cells seeded on coverslips were fixed with cold methanol for 4 min at −20 °C or with 4% of paraformaldehyde for 20 min at room temperature followed by 0.1% Triton X-100 to allow for permeabilization. Cells were then incubated with the following primary antibodies: anti-αSMA mouse monoclonal antibody (1:400) (Sigma Aldrich, Merck KGaA), anti-vimentin rabbit antibody (1:400) (AbCam, UK), and anti-fibronectin mouse monoclonal antibody (1:100) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). The primary antibodies were visualized using goat anti-rabbit Alexa Fluor 555 conjugate and goat anti-mouse Alexa Fluor 488 conjugate antibodies (1:800) (Cell Signaling Technology). Coverslips were then mounted using ProLong Gold antifade reagent with DAPI (InVitrogen, Life Technologies Corporation, Eugene, OR, USA). The fluorescence signals were analyzed by recording stained images using a CCD camera (Zeiss).

Frozen sections of rat eyes from the intracameral injection group were used for immunofluorescence staining of α-smooth muscle actin (α-SMA) to localize ciliary muscle. They were fixed in 4% paraformaldehyde for 10 min and blocked with normal goat serum at room temperature for 1 h. This was followed by incubation with anti-α-SMA mouse monoclonal antibody (1 : 100) (Sigma-Aldrich, St. Louis, USA) at 4°C overnight and subsequent incubation with fluorescein isothiocyanate- (FITC-) labeled goat anti-mouse IgG (1 : 50) (Sigma-Aldrich, St. Louis, USA) at room temperature for 2 h. Finally, the sections were incubated with DAPI nuclear staining solution at room temperature for 5 min, mounted with an antifluorescent quenching agent (Beyotime Institute of Biotechnology, Shanghai, China) and observed under a fluorescence microscope.