Mouse monoclonal antibody to α smooth muscle cell actin

Histological analysis was performed on both the ischemic and non-ischemic limbs at day 21 post hind limb ischemia surgery. Mice were euthanized and the tissue was perfused with saline followed by 10% buffered formalin for fixation. The bone was then demineralized in a formic acid based solution (Cal-Ex II, Fisher Scientific) for 48 hours before processing and paraffin embedding. Sections (5 μm thick) were immunostained for vascular smooth muscle cells or endothelial cells. Enzyme treatment was performed in proteinase K (Biolabs 2ug/ml) prior to incubation with primary antibodies. For smooth muscle cells, the sections were stained using a mouse monoclonal antibody to α-smooth muscle cell actin (Sigma) as the primary antibody detected using the avidin-biotin-alkaline phosphatase method (Vectastain ABC-AP, Vector Laboratories) with a hematoxylin counter-stain. Images of the entire section were acquired used the Hamamatsu NanoZoomer SQ slide scanner. To visualize endothelial cells slides were stained with biotinylated Lectin antibody (Biotinylated Griffonia simplifolia Lectin 1, Vector lab), followed by incubation with Streptavidin Qdot 655 (Invitrogen). Images were acquired using the 20X Plan-Neo air objective on a Zeiss Axioskop microscope equipped with an AxioCam camera. ImageJ software (NIH) was used to count the number of vessels for analysis.