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CD44v6 Antibody Staining Protocol for Immunohistochemistry
Description: Mouse monoclonal antibody derived from a fusion of mouse myeloma cells (SP2/0) and spleen cells from a mouse immunized with a human chimeric fusion protein CD44v3-10-Fc. Purified from tissue culture supernate by affinity chromatography on a protein-A matrix.
Primary Antibody
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Name: CD44v6 Antibody |
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Clone: 2F10, Mouse anti-Human |
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Supplier: R&D Systems |
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Catalog Number: BBA13 |
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Dilution: 1:500 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: Cell membrane |
| Images: Search image |
Additional Information:
| Tissue Type: Colon |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Human PBMCs, colon, breast carcinoma, pancreatic adenocarcinomas |
| 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 |
Notes:
Not need to perform antigen retrieval on acetone fixed frozen sections |
References:
1. Morris SF, O'Hanlon DM, McLaughlin R, McHale T, Connolly GE, Given HF. (2001) The prognostic significance of CD44s and CD44v6 expression in stage two breast carcinoma: an immunohistochemical study. Eur J Surg Oncol. 27(6):527-31. PubMed Abstract
2. Castella EM, Ariza A, Ojanguren I, Mate JL, Roca X, Fernandez-Vasalo A, Navas-Palacios JJ (1996) Differential expression of CD44v6 in adenocarcinoma of the pancreas: an immunohistochemical study. Virchows Arch. 429(4-5):191-5. PubMed Abstract
3. Rosenberg WM, Prince C, Kaklamanis L, Fox SB, Jackson DG, Simmons DL, Chapman RW, Trowell JM, Jewell DP, Bell JI (1995) Increased expression of CD44v6 and CD44v3 in ulcerative colitis but not colonic Crohn's disease. Lancet. 345(8959):1205-9. PubMed Abstract