Corneal Tissue Staining and Cytotoxicity Assay

Hematoxylin-eosin (H&E) and Masson's trichome staining were performed using the standard procedure for visualizing morphologic details, as reported earlier.^{40 (link),45 (link)} Immunofluorescence staining was performed following reported methods^{17 (link)} to measure myofibroblasts in the corneas using antibody-specific α-smooth muscle actin (α-SMA), a marker for myofibroblasts. Briefly, corneal sections were blocked with 2% bovine serum albumin at room temperature for 30 minutes, followed by incubation with α-SMA mouse monoclonal primary antibody (1:200 dilution, M0851; Dako, Carpentaria, CA, USA) for 90 minutes and then incubated with Alexa-Fluor 488 goat anti-mouse IgG secondary antibody (1:1000 dilution, A11001; Invitrogen, Carlsbad, CA, USA) for 1 hour at room temperature. Appropriate positive and negative controls were included in each immunostaining. Quantification of α-SMA-positive cells was performed in six randomly selected, nonoverlapping, full-thickness central corneal columns, extending from the anterior stromal surface to the posterior stromal surface at 200× and 400× magnification fields.
The toxicity of PEI2-GNPs and BMP7+HGF gene therapy was determined by performing a TUNEL assay (ApopTag; Millipore, Temecula, CA, USA). Corneal sections were fixed in acetone at –20°C for 10 minutes, and a TUNEL assay was performed per the manufacturer's instructions, including suitable positive and negative controls. Rhodamine-conjugated apoptotic cells (red) and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI)–stained nuclei (blue) were viewed and photographed with a fluorescence microscope (Leica) fitted with a digital camera system (SpotCamRT KE; Diagnostic Instruments, Sterling Heights, MI, USA). DAPI-stained nuclei and TUNEL-positive cells in untreated and treated tissues were quantified at 200× and 400× magnification in six randomly selected nonoverlapping areas, as previously reported.^{17 (link),28 (link)}