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2 protocols using goat polyclonal anti cd31

1

Quantifying Capillary Density via Immunohistochemistry

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Capillary density was performed 14 days after the surgical procedure by immunohistochemistry. Mice were sacrificed, hearts were removed, and paraffin-embedded sections were prepared. Endothelial cells were labeled using goat polyclonal anti-CD31 (Santa Cruz Biotechnology, Santa Cruz, CA) followed by a biotinylated horse anti-goat secondary antibody (Vector Laboratories). The reaction product (brown) was visualized with 3,3′-diaminobenzidine substrate using the Vector ABC Vectastain Elite Kit (Vector Laboratories, Burlingame, CA). Images were captured and stored in digital Tiff file format for later image analysis. Counts of capillary and arteriolar density per square millimeter were obtained after superimposing a calibrated morphometric grid on each digital image using Adobe Photoshop Software.
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2

Immunohistochemical Staining Protocol

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The following primary antibodies were used: goat polyclonal anti-AQP4 (Santa Cruz, CA, USA); goat polyclonal anti-CD31 (Santa Cruz, CA, USA); rabbit polyclonal anti-GFAP (Sigma-Aldrich, Milan, Italy); mouse monoclonal anti-GFAP (clone G-A-5, Millipore, Merck KGaA, Darmstadt, Germany); mouse monoclonal anti-GAPDH (MAB 374, Millipore, Merck KGaA, Darmstadt, Germany); mouse monoclonal anti-Glutamine Synthetase (MAB 302, Millipore, Merck); goat polyclonal anti-actin (Santa Cruz, CA, USA). The following secondary antibodies (all from Invitrogen, Milan, Italy) were used for immunofluorescence: donkey anti-goat Alexa Fluor488; donkey anti-mouse Alexa Fluor488; donkey anti-rabbit Alexa Fluor594; donkey anti-goat Alexa Fluor594; donkey anti-rabbit Alexa Fluor647. The following secondary antibodies (all from Santa Cruz, CA, USA) were used for Western blot:
goat anti-mouse IgG-horseradish peroxidase (HRP); goat anti-rabbit IgG-HRP; donkey anti-goat IgG-HRP.
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