Mouse anti human cd45

IF double staining protocol was used in order to determine the relative expression of CD90 with CD24 and also the expression difference between CD90 and CD45 or α-SMA. Briefly, the sections were placed in antigen retrieval citrate solution heated at 92–98°C for 15 minutes. Then these sections were blocked with 2% BSA at room temperature. The primary antibodies used were anti-human CD90 with anti-human CD24, mouse anti-human α-SMA (1:400, Sigma) or mouse anti-human CD45 (1:100, Abcam, Cambridge, MA), respectively. These antibodies were added to cover sections and were incubated at 4°C overnight. DyLight 488 anti-rabbit IgG (green) and DyLight 549 anti-mouse IgG (red, Vector laboratories, Burlingame, CA) were incubated (1:150 dilution respectively) in a dark place under normal conditions for 1 hour. Nuclei visualization was performed by incubating DAPI (blue) for 15 minutes. These were washed with PBST for 15 minutes between each step. The sections were finally dehydrated and mounted.