Monoclonal mouse anti α smooth muscle actin antibody

Lung tissue samples (5 μm) of right lobe was removed from the rats. The tissue samples were fixed with 10% neutral buffered formalin (Guangzhou Whiga Technology Co., Ltd.) and the specimens were dehydrated (by incubating sequentially for 2 h in 75%, 85%, 95% and 100% alcohol, in turn) and embedded in paraffin (Guangzhou Whiga Technology Co., Ltd.). Fixed embedded tissue sections (size, 5 μm) were sliced, placed on glass slides and deparaffinized. Subsequently, α-smooth muscle actin immunohistochemical staining with monoclonal mouse anti-α-smooth muscle actin antibody (1:100; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to determine smooth muscle cells in the lung. The ASM areas were stained brown as a result of the α-smooth muscle actin immunohistochemical staining. The ASM areas (μm²) of eight independent bronchioles were measured using Image-Pro Plus version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). The results are expressed as a proportion of the total airway area [i.e. the lumen plus the airway wall for the external area at the adventitial border (μm²)].

ASMCs were identified by α-smooth muscle actin immunocytochemical staining, as previously described (18 (link),19 (link)). Briefly, ASMCs were seeded onto sterile glass coverslips and grown to 70% confluence. Cells were fixed in 4% paraformaldehyde for 20 min, and the sections were reacted with 3% H₂O₂ (Guangzhou Whiga Technology Co., Ltd.) After a rinse in phosphate-buffered saline (PBS), the sections were blocked with 2% bovine serum albumin (BSA; Guangzhou Whiga Technology Co., Ltd.) and incubated with monoclonal mouse anti-α-smooth muscle actin antibody (1:300; Thermo Fisher Scientific, Inc.) at 4°C overnight for immunolabeling. Subsequently, the sections were incubated with a secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:300; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 37°C and then washed three times with PBS. The peroxidase activity was visualized by a color reaction using 3,3′-diaminobenzidine (1 ml; Wuhan Boster Biological Technology, Ltd., Wuhan, China) as the substrate. The slides were then counterstained with hematoxylin (Wuhan Boster Biological Technology, Ltd.), mounted and examined under a microscope (Olympus Corp., Tokyo, Japan) using AxioVision 4.1 software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).