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Epidermal Growth Factor Receptor (EGFr) Antibody Staining Protocol for Immunohistochemistry
Description: The epidermal growth factor receptor (EGFr, HER-1, c-erbB-1) is a 170 kDa transmembrane protein consisting of an extracellular EGF-binding domain, a short transmembrane region, and an intracellular domain with ligand-activated tyrosine kinase activity. EGFr is expressed in many normal epithelial tissues, particularly in the basal layers of stratified epithelium and in squamous epithelium. In normal tissues, Zymed’s monoclonal anti-EGFr (clone: 31G7) stains epidermal cells of skin, esophagus, kidney, testis, placenta, and prostate. Positive staining appears as a linear to finely granular pattern in the cell membrane and adjacent cytoplasm, or as coarsely granular cytoplasmic staining.
Over-expression of EGFr has been observed in many types of neoplasia, as the result of gene amplification and/or increased protein transcription. EGFr over-expression has been detected in endometrial carcinoma, correlated with myometrial invasion, in lung neoplasms including metaplastic squamous epithelium, squamous cell carcinoma, adenocarcinoma, and neuroendocrine lung tumors, and in glioblastoma multiforme brain tumors. EGFr expression has also been reported in most head and neck carcinomas. Variable EGFr expression has been positively correlated with increasing tumor stage in transitional bladder, renal cell, and gastric carcinomas, and in inverse correlation to estrogen receptor (ER)-negative tumor status in breast carcinoma.
Primary Antibody
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Name: EGFr Antibody |
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Clone: 31G7, Mouse anti-Human |
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Supplier: Zymed |
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Catalog Number: 28-0005 |
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Dilution: 1:20 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 minutes/room temperature |
Antigen Retrieval
| Device: Incubator |
| Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101) |
| Heat/Cool Temperature: 37 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:
| Tissue Type: Skin |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Skin, esophagus, kidney, testis, placenta, and prostate, breast carcinoma, lung tumors, brain tumors. |
| Negative Control: Omit primary antibody, isotype control or 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 |
Notes:
No need to perform antigen retrieval on frozen sections |
References: