Alpha smooth muscle actin α sma

Masson's trichrome staining and immunohistochemistry were performed as previously described.23 (link) The midshaft of the penis was immediately fixed in 4% paraformaldehyde overnight and then embedded in paraffin for further histologic studies. To assess fibrosis, Masson's trichrome staining was used to evaluate the smooth muscle-to-collagen ratio using images at ×200 magnification. Alpha smooth muscle actin (α-SMA; 1:50; Cell Signaling Technology, Danvers, MA, USA) was used to assess smooth muscle content at ×200 magnifications. For apoptosis, the tissue sections were incubated with antibody to caspase-3 (1:200; Cell Signaling Technology) at ×200 magnification. To assess oxidative stress, the tissue sections were incubated with antibodies to nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) (both 1:100; Cell Signaling Technology) at ×200 magnification. Ten sections of the corpus cavernosum were evaluated for each group, and six fields were counted for each section at ×200 magnification on a fluorescence microscope (Olympus, Tokyo, Japan) by an observer blinded to group allocation. Images were analyzed using Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA). The expression levels were standardized as a ratio to control.

Antibodies against Nrf2 (Cat# 12712), NF-κB p65 (Cat# 3034), collagen A1 (Cat# 66948), collagen 3A (Cat#30565), alpha-smooth muscle actin (α-SMA) (Cat#19245), β-actin (Cat# 4970), and Lamin A (Cat# 86846) were purchased from Cell Signalling, USA. Antibodies against TGFβ-1 (Cat# sc-130348), smad3 (Cat# sc-101154), smad7 (Cat# sc-365846), phospho-smad3 (Ser⁴²⁵) (Cat# sc-517575), and BMP7 (Cat# sc-53917) were purchased from Santa Cruze biotechnology, USA. A Bradford-based protein assay kit (Cat# 23200), phosphate buffer saline solution (Cat# 20012043), protease inhibitor cocktail (Cat# A32965), radio-immuno-precipitation assay RIPA buffer (RIPA) buffer (Cat# ab156-34); and nuclear-cytoplasmic/nuclear extraction kit (Cat# 78833) were purchased from ThermoFisher scientific.

Liver tissues were embedded in paraffin and stained with hematoxylin & eosin (H&E) or Sirius Red. Liver tissues were embedded in optimum cutting temperature (OCT) medium and frozen. Then, frozen sections were prepared and stained with Oil Red O. Immunohistochemical staining for F4/80 (70076; Cell Signaling Technology, Danvers, MA, USA), and alpha-smooth muscle actin (α-SMA; 19245; Cell Signaling Technology) was performed on the liver sections. Images were acquired with a light microscope (Olympus, Tokyo, Japan).