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WT1 Antibody Staining Protocol for Immunohistochemistry
Description: Wilms’ tumor (WT) is an embryonal malignancy of the kidney that affects 1 in 10,000 infants and, like retinoblastoma, is observed in both sporadic and inherited forms. The Wilms’ tumor locus has been mapped at chromosome 11p13 as a tumor suppressor gene which encodes a DNA binding protein with four zinc fingers and a glutamine-proline rich amino terminus. The Wilms’ tumor protein binds the DNA sequence GCGGGGGCG, a recognition element common to the early growth response (Egr) family of Zn++ finger transcriptional activators. However, in contrast to Egr transcription factors, WT1 behaves as a transcriptional repressor in transient transfection assays with synthetic promotor constructs.
Primary Antibody
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Name: WT1 Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Santa Cruz Biotechnology |
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Catalog Number: sc-192 |
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Dilution: 1:2000 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: Nuclear in kidney glomeruli |
| Images: Search image |
Additional Information:
| Species Reactivity: Human, mouse, rat |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: 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: