Immunohistochemical Analysis of Carotid Plaque

Tissue samples were obtained from surgically resected carotid atherosclerotic plaque. The samples were fixed with formalin and embedded in paraffin. The samples were pretreated by heating them in a microwave in a citrate buffer for 10 min at 500 W. The first antibodies used were the monoclonal anti-human RPA2 antibody (Santa Cruz Biotechnology), the anti-CD68 antibody (Santa Cruz) and the anti-SMC (smooth muscle cells) antibody (Santa Cruz) at a dilution of 1:100. After incubation at 37°C for 60 min, the specimens were incubated with biotin-labeled rabbit anti-mouse-IgG secondary antibody and, next, with streptavidin-labeled peroxidase. Sections were counter-stained with hematoxylin after the DAB reaction as described in the literature [12 (link)].

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Machida T., Kubota M., Kobayashi E., Iwadate Y., Saeki N., Yamaura A., Nomura F., Takiguchi M, & Hiwasa T. (2015). Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX). Journal of Translational Medicine, 13, 71.

Publication 2015

anti-CD68 antibody

Anti antibody Anti igg Antibodies Antibody Atherosclerotic plaque Biotin Buffer Carotid Citrate Dilution Embedded paraffin Formalin Human Microwave Mouse Peroxidase Rabbit Rpa2 Smooth muscle cells Streptavidin Surgically Tissue

Corresponding Organization :

Other organizations : Chiba Cerebral and Cardiovascular Center, Kameda Medical Center, Chiba University, Chiba Prefectural University of Health Sciences

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Pretreatment by heating the tissue samples in a microwave in a citrate buffer for 10 min at 500 W

dependent variables

Expression of RPA2 protein
Expression of CD68 protein
Expression of smooth muscle cell (SMC) protein

control variables

Tissue samples obtained from surgically resected carotid atherosclerotic plaque
Tissue samples fixed with formalin and embedded in paraffin
Primary antibody dilution of 1:100
Incubation time of 60 min at 37°C
Use of biotin-labeled rabbit anti-mouse-IgG secondary antibody
Use of streptavidin-labeled peroxidase
Counterstaining with hematoxylin after the DAB reaction

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