Anti α smooth muscle actin antibody

Keloids and unaffected normal skin tissue around the keloids were obtained surgically with the patient’s informed consent. Ten tissue samples were taken from keloids located on the shoulder, chest, or abdomen of 8 individuals (Table 1: K1–K10, male:female ratio, 2:3; mean age, 36.2; Fig. 1). This protocol was approved by the Keio University School of Medicine institutional review board.
Excised skin specimens were fixed with 4% paraformaldehyde, and paraffin sections (4 μm) were taken. Slides were stained with anti-YAP/TAZ signaling antibody (1:100 dilution, Cell Signaling Technology) and anti-αsmooth muscle actin (SMA) antibody (1:100 dilution; Dako, Santa Clara, Calif.). Fluorescent images were obtained using a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan).
To analyze YAP/TAZ localization in fibroblasts, keratinocytes, and endothelial cells from keloids and unaffected regions, the YAP/TAZ nucleus–positive cell index (percentage of cells with YAP/TAZ localized to the nucleus) was calculated by counting the number of immunoreactive cells and total cells in the keloid and unaffected areas (n = 4 per group). Cell counting was performed by an independent observer blinded to the sample identity. Statistical differences were determined using a nonparametric Mann-Whitney U test. P values less than 0.05 were considered significant.