Anti αsma mouse igg2a

Cells were fixed in 4% PFA and stained with the following primary antibodies: anti- β-III tubulin (mouse IgG2b, 1:500, Sigma T8660), anti- αSMA (mouse IgG2a, 1:1000, Sigma A2547), and anti-GFAP (rat IgG, 1:500, Invitrogen 13-0300). The secondary antibodies used for staining were the following: anti-mouse IgG2b (Alexa 488 A-21141), anti-mouse IgG2a (Alexa 350 A-21130), and anti-rat IgG (Alexa 555 A21434). The stained samples were observed under a universal fluorescence microscope (Axioskop 2 Plus; Carl Zeiss).

The ASM cells and fibroblasts were characterized by flow cytometry before and at 31 days of recellularization (transfer day), as described previously (27 (link)). Briefly, the cells were stained for intracellular proteins with anti–α-SMA (mouse IgG2a, 1/250; Sigma Aldrich, St. Louis, MO, United States) and anti-desmin (rabbit polyclonal IgG, 1/200; Abcam, Cambridge, United Kingdom,) antibodies for 1 h. The cells were then washed three times and incubated for 30 min in the dark with fluorescent dye–conjugated anti-IgG antibodies. Isotype-matched control antibodies (mouse IgG2a and rabbit IgG) were used as negative control. All signals greater than those of the isotype-matched control antibodies were considered positive, and the degree of staining was evaluated as the mean fluorescence intensity and mean percentage of positive cells for both α-SMA and desmin.