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

Name: WT1 Antibody

Clone: Rabbit polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-192

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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