Rabbit anti laminin

At day 7 after stroke, rats were given an overdose of ketamine/xylazine and then transcardially perfused with cold saline followed by 10% buffer-formalin via the ascending aorta. The brains were removed and postfixed in 10% buffer-formalin (Fischer Scientific) for 48 h and then stored at 4°C in a solution of 30% sucrose–saline for 2 days. The brains were embedded in OCT and sectioned coronally in 12-μm-thick slices starting from the frontal pole at an interval of 2 mm. Primary antibodies were incubated overnight at 4°C at the following dilutions: rabbit anti-laminin (1/200; Dako Cytomation, Carpinteria, California, USA). After washing, slides were incubated with fluorescent secondary antibodies, cover-slipped with Vectashield mounting medium (Vector Laboratories, Burlingame, California, USA) and viewed using ZeisAxio Observer.Z1 fluorescent microscope. Negative controls were prepared by omitting the primary antibodies. Laminin-stained vascular profiles (including arterioles and venules) were quantified using ImageJ software (NIH) in 3 different fields per section digitized from the ischemic border zone using a 10X objective lens. An average of the 3 fields of interest (FOI) per animal was calculated and plotted.