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Description: CD31 is a transmembrane glycoprotein, 130-140 kDa, also know as platelet-endothelium cell adhesion molecule (PECAM-1). CD31 is ligand for CD38 and plays a role in thrombosis and angiogenesis. CD31 is strongly expressed in endothelial cells and weakly expressed in megakaryocytes, platelets, occasional plasma cells, lymphocytes (espc. marginal zone B-cells, peripheral T-cells) and neutrophils. JC/70A reacts with a glycoprotein of about 100 kDa in endothelial cells and 130 kDa in platelets and can be used for the immunohistochemical detection of normal and tumor tissues.
Fixation: Formalin-fixed, paraffin embedded sections, acetone fixed frozen sections, cell smears.
Positive Controls: Tonsil, skin.
Solutions and Reagents:
Primary Antibody:
Mouse Anti-Human CD31 (Clone JC/70A) (DakoCytomation, Cat# M0823). Optimal dilution 1:50. Species Reactivity: Human (refer to antibody datasheet for more information).
Secondary Antibody:
Horse Anti-Mouse IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-2000). Optimal dilution 1:500.
Detection Reagent:
HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal dilution 1:500
Procedure:
1. Paraffin sections to distilled water (refer to general IHC protocols as needed).
2. Epitope Retrieval: Use Tris-EDTA Buffer Epitope Retrieval Method. Briefly, pre-heat steamer or water bath with staining dish containing Tris-EDTA Buffer (pH 9.0) until temperature reaches 95-100 °C. Immerse slides in the staining dish and place the lid loosely on the staining dish. Incubate for 20 minutes and turn off the steamer or water bath. Place the staining dish at room temperature and allow the slides to cool for 20 minutes.
3. Rinse sections in 2 changes of washing buffer, 2 minutes each.
4. Serum Blocking: incubate sections with normal horse serum blocking solution for 30 minutes to block non-specific binding of immunoglobulin.
5. Primary Antibody: incubate sections with Mouse Anti-Human CD31 (Clone JC/70A) (DakoCytomation, Cat# M0823) diluted 1:50 in primary antibody dilution buffer for 1 hour at room temperature.
6. Rinse in washing buffer for 2x2min.
7. Peroxidase Blocking: incubate sections in peroxidase blocking solution for 10 minutes to block endogenous peroxidase activity.
8. Rinse in washing buffer for 3x2 min.
9. Secondary Antibody: incubate sections with biotinylated Horse Anti-Mouse IgG diluted in secondary antibody dilution buffer for 30 minutes at room temperature.
10. Rinse in washing buffer for 3x2min.
11. Detection: incubate sections with HRP-Streptavidin diluted in HRP-streptavidin dilution buffer for 30 minutes at room temperature.
12. Rinse in washing buffer for 3x2min.
13. Chromogen/Substrate: incubate sections in DAB peroxidase substrate solution for 5-10 minutes.
14. Rinse in distilled water briefly.
15. Counterstain with Gill's hematoxylin solution or Mayer's hematoxylin solution if desired.
16. Rinse in running tap water for 5 minutes.
17. Dehydrate through 95% ethanol for 2 minutes, 100% ethanol for 2x3min.
18. Clear in xylene for 2x3min.
19. Coverslip with permanent mounting medium.
Results:
Staining pattern: Membrane/cytoplasmic of endothelial cells (Search Images)
Notes:
1. Related Protocols: PECAM-1 (M-20) (Santa Cruz Biotechnology)
CD31 (PECAM-1) (Pharmingen)
2. Avidin/Biotin Blocking may be needed to block endogenous biotin activity for certain tissues such as kidney, liver, prostate, colon and gut, which may contain endogenous biotin.
3. For frozen sections, snap frozen fresh tissues in isopentane pre-cooled in liquid nitrogen, embedded in OCT compound in cryomolds. Cut 4-8 um cryostat sections and mount on superfrost plus slides. Store slides at - 80°C until needed. Before staining, air dry slides at room temperature for 30 minutes and fix in ice-cold acetone for 5 minutes. Air dry for another 30 minutes. Then start from step 3 for routine immunostaining.
4. This antibody does not have cross reactivity with mouse and rat. |
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发表于 2007-9-20 00:21:13