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Alpha-Gal (a-Gal) Epitope Antibody Staining Protocol for Immunohistochemistry
Description: Widely described antibody against α-galactose residues with broad cross-reactivity among different species. Useful for measuring the α-Gal epitope expression on cells, glycolipids and glycoproteins, characterization of hyperacute rejection (HAR) in organ and tissue transplantations and monitoring xenotransplantation experiments. The IgM isotype mimicks in vivo reactions and can be used for cytotoxicity assays for α-Gal specific pathways (addition or presence of complement necessary). Ideal as a high-throughput screening tool for inhibitors of antibody induced cytotoxicity.
Organ transplantation from pig to human result in HAR. Humans
naturally produce large quantities of anti-α-Gal antibodies, which
represent 1-3% of all circulating immunoglobulins and are produced
by about 1% of all B cells. When pig organs or tissues are
transplanted into the human body, the IgM isotype of anti-Gal binds
to α-Gal epitopes, which causes activation of the complement
cascade, resulting in cell lysis. This rapid activation of
complement by anti-Gal IgM is an immunological barrier that poses
the greatest risk of initiating HAR. The monoclonal antibody M86 to
Galα1-3Gal epitopes developed by U. Galili, et al. is an
essential tool in the study of human xenotransplantation and related
research.
Primary Antibody
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Name: alpha-Gal (a-Gal) Epitope Antibody |
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Clone: M86, Mouse Monoclonal |
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Supplier: Alexis Biochemicals |
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Catalog Number: ALX-801-090 |
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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: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102) |
| Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) |
| Heat/Cool Temperature: 95-100 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/Cytoplasmic |
| Images: Search image |
Additional Information:
| Species Reactivity: Human |
| Fixation: Formalin-fixed paraffin sections |
| Positive Control: Cardiac tissues, heart |
| 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 |
References:
1. A sensitive assay for measuring
alpha-Gal epitope expression on cells by a monoclonal anti-Gal
antibody: U. Galili, et al.; Transplantation 65, 1129 (1998).
2. Differential expression of alpha-Gal epitopes on pig and mouse
organs: M. Tanemura & U. Galili; Transplant. Proc. 32, 843
(2000).