Cd31 antibody

Macrophages and neutrophils were stained with alpha-napthyl acetate esterase (Sigma-Aldrich, Germany) or naphthol as-d chloroacetate (Sigma-Aldrich, Germany), respectively. Each heart was analyzed on the basis of either 3 or 4 sections. Cells were counted in either 3 or 4 fields of each section, respectively. Stained cells (macrophages in yellow-brown, neutrophils in red) were counted per mm² of the infarct area. We analyzed neoangiogenesis in infracted areas by counting CD31-positive ring structures on the basis of CD31 antibody (Santa Cruz Biotechnology, Heidelberg, Germany). These values are expressed as an absolute number per mm². Nuclei undergoing apoptosis were stained with MEBSTAIN apoptosis kit II (MBL International, Woburn, MA, USA) and TUNEL-positive (i.e. apoptotic) nuclei were counted. The relevant values were calculated as a percentage of all nuclei (apoptotic index). The collagen content of the infarct-regions was determined using Gomori’s 1-step trichrome staining. The collagen blue-stained areas were measured by computer assisted planimetry (P-Cell Software, Olympus) and expressed as percent of the total infarct area.

The equipment used for generating electrical burns was as follows: the adjustable voltage regulator (0-250V; rated power, 2000W), the power supply switch, the microcomputer time-controlled switch (accurate to 1 sec), the current meter (range 0-600mA; accuracy, 1mA), the voltage meter (range 0-450V; accuracy, 10V), two copper electrode clips, and two copper electrode plates (3mm×3mm), were purchased from Delixi Electric (Zhejiang, China). The CD31 antibody and the donkey anti-goat secondary antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The ROS fluorescence detection kit was purchased from Genmed Pharmaceutical Technology (Shanghai, China). First Strand cDNA Synthesis Kit was purchased from Takara Bio (Dalian, China). PCR primers were synthesized by Sangon Biotech (Shanghai, China). ELISA kit of SDF-1α was obtained from Abnova (Taipei, Taiwan). Cell culture reagents were purchased from Invitrogen (Carlsbad, CA, USA). If not stated otherwise, reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Vessels were visualized by CD31 staining at 56 days after MCAO. Free floating sections were prepared from fixed and dehydrated brains and stained with CD31 antibody (1:500, Santa Cruz Biotechnology). After incubation in primary antibody for 1 hour at room temperature and overnight at 4°C, coronal brain sections were incubated with 488-conjugated goat anti-rabbit (1:1000, Jackson ImmunoResearch Labortories, Inc) secondary antibodies to stain vessels. Four sections at 0.1 mm intervals were analyzed in each brain. The number of CD31⁺ vessels longer than 50 μm was counted in the ipsilateral peri-infarct striatum. The vascular length per mm² was calculated using ImageJ software by a blinded observer.

Tumors were harvested in 4% paraformaldehyde for 24 h and embedded in paraffin. For immunostaining, 5-μm deparaffinized poly-L-lysine-coated sections were treated with 3% hydrogen peroxide for non-specific binding. A CD31 antibody (Santa Cruz Biotechnology) diluted at 1∶200 was used for immunohistochemical staining by the peroxidase-antiperoxidase technique. Brown staining indicated a positive result.
MVD was expressed as the number of microvessels per field. A single microvessel was defined as any CD31-immunostained endothelial cell that was separated from adjacent tumor cells and other connective tissue elements. Large vessels with thick muscular walls were not counted, and the presence of a lumen was not necessary for scoring as a microvessel. In each sample, the three most intense areas of neovascularization were identified and counted at low power (magnification, 100×). Average vessel counts of the three selected fields were recorded.