RAGE Antibody Staining Protocol for Immunohistochemistry


Description: RAGE is a member of the immunoglobulin superfamily of cell surface molecules that binds molecules that have been irreversibly modified by non-enzymatic glycation and oxidation, and are know as advanced glycation end products (AGEs).  It is expressed by endothelium, mononuclear phagocytes, neurons and smooth muscle cells.  Whereas RAGE is present at high levels during development, especially in the central nervous system, its levels decline during maturity. The increased expression of RAGE is associated with several pathological states, such as diabetic vasculopathy, neuropathy, retinopathy and neuropathy, and other disorders, including Alzheimer’s disease and immune/inflammatory reactions of the vessel walls.  In diabetic tissues, the production of RAGE is due to the overproduction of AGEs that eventually overwhelm the protective properties of RAGE.  This results in oxidative stress and endothelial cell dysfunction that leads to vascular disease in diabetics.  In the brain, RAGE also binds amyloid beta (AE).  Because AE is overproduced in neurons and vessels in the brains of Alzheimer disease, this leads to the hyperstimulation of RAGE.  The RAGE-AE interaction is thought to result in oxidative stress leading to neuronal degeneration.


Primary Antibody

Name: Receptor for Advanced Glycosylation End Products (RAGE-N Terminal Antiserum) Antibody

Clone: Goat polyclonal

Supplier: Research Diagnostic (RDI)

Catalog Number: RDI-RAGEabG

Dilution: 1:400 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: Cytoplasmic
Images: Search image

Additional Information:
Species Reactivity: Human
Fixation: Formalin fixed paraffin sections
Positive Control: Diabetic kidney, Alzheimer’s brain
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