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Paxillin Antibody Staining Protocol for Immunohistochemistry
Description: Paxillin is a cytoskeletal protein that is integral to the formation of focal adhesion complexes. Paxillin helps regulate gene transcription by interacting with transcription factors, however, the misregulation of nuclear functions of paxillin proteins appears to be associated with pathogenic effects. A recent study indicated that paxillin overexpression was a reporter of high HER2/neu activity. Paxillin may become a key target to aid in the monitoring of aggressive breast cancers. Paxillin may also be useful for distinguishing chromophobe renal cell carcinoma and oncocytomoa from conventional or papillary renal cell carcinoma.
Primary Antibody
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Name: Paxillin Antibody |
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Clone: ZO35, Mouse anti-Human |
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Supplier: Zymed Labs |
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Catalog Number: 18-0407 |
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Dilution: 1:100 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: Cellular membrane/cytoplasmic |
| Images: Search image |
Additional Information:
| Species Reactivity: Human |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Breast cancer/normal kidney |
| 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. Yuminamochi T, Yatomi Y, Osada M, Ohmori T, Ishii Y, Nakazawa K, Hosogaya S, Ozaki Y (2003) Expression of the LIM proteins paxillin and Hic-5 in human tissues. J Histochem Cytochem. 51(4):513-21.
2. Kuroda N, Guo L, Toi M, Naruse K, Miyazaki E, Hayashi Y, Yoshikawa C, Ashida S, Shuin T, Enzan H (2001) Paxillin: application of immunohistochemistry to the diagnosis of chromophobe renal cell carcinoma and oncocytoma. Appl Immunohistochem Mol Morphol. 9(4):315-8. PubMed Abstract
3. Salgia R, Li JL, Ewaniuk DS, Wang YB, Sattler M, Chen WC, Richards W, Pisick E, Shapiro GI, Rollins BJ, Chen LB, Griffin JD, Sugarbaker DJ (1999) Expression of the focal adhesion protein paxillin in lung cancer and its relation to cell motility. Oncogene. 18(1):67-77 PubMed Abstract