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AGE (Advanced Glycation End Products) Antibody Staining Protocol for Immunohistochemistry
Description: The non enzymatic reaction of reducing carbohydrates with lysine side chains and N terminal amino groups of macromolecules (amino acids, proteins, phospholipids and nucleic acids) is called the Maillard reaction or glycation. The latter products of this process, termed advanced glycation end products (AGEs), adversely affect the functional properties of proteins, lipids and DNA. In long lived tissue proteins, these chemical modifications accumulate with age and may contribute to the pathophysiology of aging and long term complications of diabetes, atherosclerosis and renal failure.
Primary Antibody
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Name: AGE Antibody |
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Clone: Rabbit Polyclonal |
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Supplier: Abcam |
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Catalog Number: ab23720 |
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Dilution: 1:100 - 1:200 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: Cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Kidney |
| Fixation: Formalin-fixed paraffin sections |
| Positive Control: Human lens, diabetic kidney/heart/spleen/trachea/brain, arteriosclerotic plaques. |
| 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. Kimura T et al. Accumulation of advanced
glycation end products of the Maillard reaction with age in human
hippocampal neurons. Neurosci Lett 208:53-6 (1996).
PubMed: 8731173
2. Kume S et al. Immunohistochemical and ultrastructural
detection of advanced glycation end products in atherosclerotic
lesions of human aorta with a novel specific monoclonal antibody.
Am J Pathol 147:654-67 (1995).
PubMed: 7545874
3. Miyata T et al. beta 2-Microglobulin modified with
advanced glycation end products is a major component of hemodialysis-associated
amyloidosis. J Clin Invest 92:1243-52 (1993).
PubMed: 8376584
4. Araki N et al. Immunochemical evidence for the presence of
advanced glycation end products in human lens proteins and its
positive correlation with aging. J Biol Chem 267:10211-4
(1992).
PubMed: 1587810
5. Horiuchi S et al. Immunochemical approach to characterize
advanced glycation end products of the Maillard reaction. Evidence
for the presence of a common structure. J Biol Chem 266:7329-32
(1991).
PubMed: 2019568