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CD31 Antibody Staining Protocol for Immunohistochemistry
Description: This antibody reacts with CD31, also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1). CD31 is a 130 kDa integral membrane protein, a member of the immunoglobulin superfamily, that mediates cell-to-cell adhesion. CD31 is expressed constitutively on the surface of adult and embryonic endothelial cells and is weakly expressed on many peripheral leukocytes and platelets. It has also been detected on bone marrow-derived hematopoietic stem cells and embryonic stem cells. CD31 is involved in the transendothelial emigration of neutrophils, and neutrophil PECAM-1 appears to be down-regulated after extravasation into inflamed tissues. Multiple alternatively spliced isoforms are detected during early post-implantation embryonic development; this alternative splicing is involved in the regulation of ligand specificity. CD38 and vitronectin receptor (αvβ3 integrin, CD51/CD61) are proposed to be ligands for CD31. CD31 mediated endothelial cell-cell interactions are involved in angiogenesis. The MEC13.3 mAb inhibits a variety of in vitro and in vivo functions mediated by CD31.
Primary Antibody
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Name: Rat Anti-Mouse CD31 (PECAM-1) Antibody |
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Clone: PECAM-1 |
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Supplier: BD Pharmingen |
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Catalog Number: 550274, 553370, 557355 |
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Dilution: 1:50 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed. |
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Incubation Time/Temp: 60 min/room temperature |
Antigen Retrieval
| Device: Incubator |
| Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101) (not necessary for zinc fixed paraffin section) |
| Heat/Cool Temperature: 37 ºC/room temperature |
| Heat/Cool Time: 20 minutes/20 minutes |
Detection Methods
| Standard Method: ABC Method or LSAB Method |
| Enhanced Method: Polymeric Methods |
Chromogen Substrate
| Reagent: DAB |
| Incubation Time/Temperature: 1-3 minutes/room temperature |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Membrane of endothelial cells |
| Images: Search image |
Additional Information:
| Tissue Type: Mouse kidney |
| Fixation: Zinc fixative fixed paraffin sections, acetone fixed frozen sections |
| Positive Control: Tonsil, skin, liver, kidney |
| Negative Control: Omit primary antibody, isotype control, absorption control |
| Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary |
Notes:
There is no need to perform antigen retrieval on acetone fixed frozen sections |
References:
1. Huss WJ, et al (2001) Angiogenesis and prostate cancer: Identification of a molecular progression switch. Cancer Res. 61:2736-2743.
2.
Vecchi A,
et al (1994) Monoclonal antibodies specific for endothelial cells of
mouse blood vessels. Their application in the identification of
adult and embryonic endothelium. Eur. J. Cell Biol. 63:247-254.
3. Vanzulli S, et al (1997) Detection of endothelial cells by MEC 13.3 monoclonal antibody in mice mammary tumors. Biocell 21:39-46.
4. Delisser HM, et al (1997) Involvement of endothelial PECAM-1/CD31 in angiogenesis. Am. J. Pathol. 151:671-677.