Ultra cruz mounting medium containing dapi

Immunofluorescence staining was performed as we described previously [38 (link), 41 (link), 42 (link)]. Briefly, cells were seeded with glass coverslips into 6-well plates and transfected with a constructed pAAV-SklF6H vector for 2 days. At the end of culturing, cells were washed in cold PBS and fixed in cold 4% paraformaldehyde for 15 minutes at room temperature. Fixed cells were washed using PBS and permeabilized by incubation with 0.25% Triton X-100 for 5 minutes. The cells were then washed in PBS, blocked using 10% BSA in PBS, and incubated with anti-FLAG M2 antibody (1:500) and ESD antibody (1:500) in incubation buffer (3% BSA in PBS) overnight at 4°C. Cells were washed in PBS and incubated with Alexa 568-conjugated (1:1,000) or Alexa 488-conjugated (1:1,000) secondary antibodies (Invitrogen) in incubation buffer for 1 hour at room temperature. The cells were then washed in PBS and mounted with Ultra Cruz Mounting Medium containing DAPI (Santa Cruz Biotechnology) and cured for at least 24 hours at room temperature in the dark. Images were taken using a Nikon Eclipse Ti confocal microscope.

The histologic and immunohistochemical analyses were performed as previously described^{46 (link),47 (link)}. In brief, sections of paraffin (5 µm thickness) embedded corneas, lens and retinas were stained with hematoxylin and eosin histochemistry and visualized under light microscopy (Axioplan 2; Carl Zeiss, Germany). For immunohistochemistry staining, sections were subjected to antigen retrieval in citrate buffer (pH = 6.0) for 20 min, and the slides were rinsed with phosphate-buffered saline (PBS). The slides were quenched with 10 mM ammonia chloride, followed by a blocking step for 1 h. They are then stained using the following primary antibodies: cellular fibronectin (Millipore, USA) diluted 1∶100; α-SMA (Dako Cytomation, Denmark) diluted 1∶50; HSP-47, Enzolife Sciences, Switzerland) diluted 1:200; CD11b (BD Pharmingen, USA) diluted 1:50, in the blocking solution. The secondary antibody was goat anti-mouse Alexa Fluor 488-conjugated (Invitrogen, USA). Slides were then mounted with UltraCruz mounting medium containing DAPI (Santa Cruz Biotechnology, USA) and were observed and imaged with a fluorescence microscope (Axioplan 2). To detect apoptotic cells, a fluorescence-based terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay (Roche Applied Science, USA) was used according to the manufacturer’s instructions.