To assess angiogenesis in the infarct border-zone, 10 μm sections taken at the midpapillary level were stained with primary antibodies such as Biotinylated Griffonia Simplicifolia Lectin I (GSL I) isolectin B4 (Vector Laboratories, B-1205-.5, 1:200), anti-alpha smooth muscle Actin antibody Alexa Fluor 594 conjugate (SMA-AF594; Abcam, ab202368, 1:300), and wheat germ agglutinin, Alexa Fluor 647 conjugate (WGA-AF647, Thermo Fisher Scientific, 1:300). Slides were then stained with secondary antibodies against native streptavidin protein conjugated with fluorescein isothiocyanate (FITC, Abcam, ab136201, 1:1000). Images were taken on the Leica DM5000b (Leica, Wetzlar, Germany) at 20x magnification using a DFC350 FX monochrome camera (Leica). Three high-power fields per animal were taken of the peri-infarct myocardium, defined as one field away from WGA-labeled scar. Images were quantified for total vasculature using ImageJ. Capillaries were identified as areas expressing isolectin B4 (I-B4) alone, while areas co-expressing I-B4 and SMA were identified as arterioles.
Ab136201
Manufactured by Abcam
Sourced in United Kingdom
Ab136201 is a primary antibody produced in rabbit. It is designed for use in immunohistochemistry and western blotting applications. The antibody specifically targets an unspecified antigen.
Automatically generated - may contain errors
2 protocols using ab136201
1
Quantifying Peri-Infarct Angiogenesis in Myocardium
2
Visualization of Internalized Biomolecules
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Following the internalization assay, the cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min, treated with permeabilization buffer (90% methanol, 5% acetic acid), and then blocked with 10% FBS in PBS for 20 min. The cells were incubated with FITC-labeled streptavidin (ab136201; Abcam, Cambridge, UK) in 10% FBS in PBS for 1 h at room temperature. After washing three times with PBS, the cells were incubated with DAPI for 5 min and mounted using VECTASHIELD Mounting Medium with DAPI (H-1200; Vector Laboratories, Burlingame, CA, USA). Images were acquired using a BZ-X700 fluorescence microscope (KEYENCE, Osaka, Japan). Image processing was performed using Adobe Photoshop CS2.
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