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

Name: alpha-Gal (a-Gal) Epitope Antibody

Clone: M86, Mouse Monoclonal

Supplier: Alexis Biochemicals

Catalog Number: ALX-801-090

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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



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).