Primary antibody against α sma

Immunohistochemical staining for alpha-SM actin (α-SMA) was performed to assess the percentage of the SM cell component (% α-SMA) using a primary antibody against α-SMA (1:100; Dako, Denmark), as described in previous investigations [17 (link), 18 (link), 22 , 23 (link)]. Seven rats from each group (two sections per rat) were analyzed. In each stained slide, we performed quantitative image analyses of × 40 magnification images, using Image-Pro-Plus 4.5 software. We analyzed ×40 magnification images of the penis comprising one-half of the corpora cavernosa, and % α-SMA in the total area of one-half of the cavernosa was measured.

Following treatment, cells or constructs were collected and processed for immunofluorescence, as previously described [33 (link),34 (link)]. In brief, cells or constructs were fixed in 4% paraformaldehyde for 10 min or 24 h respectively, placed in blocking buffer (1% bovine serum albumen [BSA] with 0.1% Triton-X [Sigma-Aldrich; St. Louis, MO, USA]) for 1 h, and incubated overnight at 4 °C with primary antibody against αSMA (Dako North America; Carpinteria, CA, USA) in blocking buffer. The next day, constructs were washed in phosphate buffered serum (PBS) and incubated overnight at 4 °C with a secondary donkey anti-mouse IgG-FITC antibody (Jackson ImmunoResearch; West Grove, PA, USA) in blocking buffer. TO-PRO-3 (Thermo Fisher Scientific) was used as a marker of cell nuclei. Constructs were washed, mounted (Vectashield: Vector Laboratories; Burlingame, CA, USA), observed, and photographed with a fluorescent microscope (Nikon E8000: MicroVideo Instruments; Avon, MA, USA) with the 20× objective. All images were acquired under identical photographic conditions for all treatment groups, and the brightness/contrast was kept constant. The median fluorescent intensity for αSMA staining was quantified by ImageJ software.