Anti α smooth muscle actin sma

Thirteen representative formalin-fixed, paraffin-embedded sections were randomly selected. 4 μm thick tissue sections were dewaxed in xylene, and rehydrated through graded alcohol. After the inhibition of the endogenous peroxidase and an antigen retrieval process, the sections were incubated with primary antibodies anti-α smooth muscle actin (SMA; mouse monoclonal, 1:12,000 dilution, Sigma) for 50 minutes at 37 °C. Primary antibodies were detected by EnVision kit (K5007, DAKO). The reaction was visualised using 3,3-diaminobenzidine (DAB). Finally, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. All thirteen sections were scored by an expert pathologist (DNR) as a percentage of SMA positive area per sample (Path SMA). Automated analysis of SMA-stained IHC images (Auto SMA) involved colour deconvolution14 (link) to split blue hematoxylin and brown SMA stain. Dichotomisation of SMA image in positive and negative areas was achieved by employing Otsu thresholding15 , a common image-processing approach to minimise intra-class variance.

At various times the eyes of mice were enucleated and briefly fixed in 4% paraformaldehyde (10 min on ice). The eyeballs were fixed in 70% methanol for at least 24 h at -20°C. Retinas were dissected in PBS and then washed with PBS three times, 10 min each. Following incubation in a blocking buffer (50% fetal calf serum, 20% normal goat serum in PBS) for 2h, the retinas were incubated with anti-collagen IV (diluted 1:250 in PBS containing 20% fetal calf serum, 20% normal goat serum) at 4°C overnight. In some cases retinas were also stained with anti-α-smooth muscle actin (SMA; Sigma). Retinas were then washed three times with PBS, 10 min each, incubated with secondary antibody Alexa 594 goat-anti-rabbit (Invitrogen; 1:500 dilution prepared in PBS containing 20% FCS, 20% NGS) for 2 hours at RT, washed four times with PBS, 30 min each, and mounted on a slide with PBS/glycerol (2vol/1vol). Retinas were viewed by fluorescence microscopy and images were captured in digital format using a Zeiss microscope (Carl Zeiss, Chester, VA). The central capillary dropout area was quantified, as a percentage of the whole retina area, from the digital images in masked fashion using Axiovision software (Carl Zeiss, Chester, VA). Quantification of vitreous neovascularization was performed as previously described by Stahl et al. [16 (link)].

Immunohistochemistry was performed on paraffin 5-μm-thick sections. Antigen retrieval was carried out by heating 20 minutes at 95 °C using EnVision LEX Target Retrieval Solution, Low pH (Dako, Santa Clara, CA). Endogen peroxidase activity was quenched by incubation in Peroxidase-Blocking Solution (Dako, Santa Clara, CA). Each incubation step was carried out at room temperature and followed by three sequential washes in TBS-T. To block nonspecific binding, tissues were incubated for 60 minutes in Protein-Block Serum-Free (Dako, Santa Clara, CA). Then, samples were incubated at room temperature 5 minutes with anti–von Willebrand factor (VWF, Dako, Santa Clara, CA, ref. GA527, prediluted) or 10 minutes with anti–α-smooth muscle actin (SMA, Sigma, St. Louis, MO, ref. A5228, 1:100), followed by incubations with Liquid DAB (Dako, Santa Clara, CA) for 10 minutes. Control immunohistochemical staining was performed following the same procedure but omitting the primary antibody. Procedures were performed in Autostainer Plus for IHC (Dako, Santa Clara, CA) and PT Link (Dako, Santa Clara, CA). Finally, slides were counterstained, dehydrated, and mounted.