Anti ki67 antibody

Slides containing sections of formalin fixed, paraffin embedded (FFPE) rat prostate tissue were steamed for 25 min (E. coli IHC) or 40 min (Ki-67, CD68) in high temperature target retrieval (HTTR) solution for antigen retrieval (Dako). Slides were then incubated with an anti-E. coli antibody (Virostat, Inc., product #1001) at a dilution of 1:2000, anti-CD68 antibody (Abcam ab125212) at 1:1000, or anti-Ki-67 antibody (Novocastra) at 1:1000 for 45 min at room temperature, followed by incubation with secondary antibody (PowerVision, Leica Microsystems) for 30 min at room temperature. Staining was visualized using 3,3’-Diamino-benzidine (Sigma FAST 3,3’-Diamino-benzidine tablets), and slides were counterstained with hematoxylin.

Rats were euthanized 2 or 4 weeks after cell transplantation. Hearts were harvested and fixed in 4% paraformaldehyde and incubated overnight in 15% sucrose solution. The tissues were embedded in OCT compound (Sakura Finetek), snap-frozen in liquid nitrogen, and sectioned at 10 – 20 µm thickness as described previously by our laboratory [28 (link)]. For capillary density measurement, four frozen sections of ischemic tissues were stained with primary biotinylated isolectin B4 (ILB4) (1:250, Vector Laboratories, Inc., Burlingame, CA) and secondary streptavidin Alexafluor 488 (1:400, Invitrogen). Five fields from four tissue sections were randomly selected, and the number of capillaries was counted in each field. Photographs were taken using fluorescent inverted microscopy or confocal microscopy. To evaluate apoptosis, TdT-mediated dUTP nick-end labeling (TUNEL) reaction was performed using a fluorescein in situ cell death detection kit (Roche-Molecular). To investigate proliferative cells, immunostaining with anti-Ki-67 antibody (Novocastra Laboratories) was performed. Details of the above procedures were described in our prior publications [28 (link), 31 (link)].