α sma clone 1a4

To investigate human common carotid arteries and the effect of coronary bare metal stents on human coronary arteries by histological and immunohistochemical examinations, 22 samples each that did not show aortitis syndrome and autoimmune disease were obtained at autopsy.
Resected tissues were fixed in 10% formalin and embedded in paraffin. The sections (3 µm) were stained with hematoxylin and eosin (H&E) and used for immunohistochemical examinations by the EnVision method (Dako Japan, Kyoto, Japan). Monoclonal anti-human CD68 (clone KP1, 1:100) and αSMA (clone 1A/4, 1:150) antibodies were obtained from Dako Japan, rabbit polyclonal antibodies to CCL22 (1:100) and monocyte chemoattractant protein (MCP)-1 (1:200) were obtained from Abcam (Tokyo, Japan), and monoclonal anti-human CD4 antibody (clone 4B12, undiluted) was obtained from Nichirei (Tokyo, Japan).
This investigation adhered to all the principles prescribed in the Declaration of Helsinki. The Ethics Committee of Experimentation, University of Occupational and Environmental Health, Japan, approved all procedures of the present study.

Right lung tissues of different ages were isolated, frozen in liquid nitrogen and stored at −80°C. Left lung tissues of different ages were isolated and perfused with ice-cold 10% formalin at a pressure of 25 cm H₂O and fixed for 24 h at 4°C. The lung tissues were imbedded in paraffin, sectioned at 4–5 μm, and processed for light microscopic immunohistochemistry as the previously described [18 (link),25 (link)]. Samples were incubated with a primary antibody against smooth-muscle-specific alpha-actin (α-SMA, clone:1A4; Dako, Denmark) overnight, followed by secondary goat antibody (HRP polymer) for 30 min at 37°C. At least eight slides from each lung were taken and then analyzed.
The percentage of pulmonary arterial medial thickness occupied by smooth muscle was determined according to our previous study [18 (link)]. Small pulmonary arteries of 50–150 μm in diameter were investigated via a systematic sampling method to evaluate random, nonoverlapping calibrated fields for each variable. The terminal bronchioles were used as an independent landmark for selecting small pulmonary arteries. We used Image Pro Plus software (Media Cybernetics, Bethesda, Maryland, USA) to make the measurements. Tissue slides were analyzed by a technician who was without knowledge of the group from which the tissue was taken.