VEGF Antibody Staining Protocol for Immunohistochemistry


Description: The onset of angiogenesis is believed to be an early event in tumorigenesis and may facilitate tumor progression and metastasis. Several growth factors with angiogenic activity have been described. These include fibroblast growth factors (FGFs), platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). VEGF is a dimeric glycoprotein with structural homology to PDGF. Several variants of VEGF have been described that arise by alternative mRNA splicing. It has been speculated that VEGF may function as a tumor angiogenesis factor in vivo because the expression pattern of VEGF is consistent with a role in embryonic angiogenesis. VEGF mRNA is formed in some primary tumors, VEGF is produced by tumor cell lines in vitro and VEGF mitogenic activity appears to be restricted to endothelial cells. A member of the PDGF receptor family, Flt, has been identified as a highaffinity receptor for VEGF.


Primary Antibody

Name: VEGF (147) Antibody

Clone: Rabbit polyclonal

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-507

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.

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) or IHC-TekTM Proteinase K Solution (Cat# IW-1101)
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: Cytoplasmic/membrane
Images: Search image

Additional Information:
Species Reactivity: Human, mouse, rat
Fixation: Formalin fixed paraffin sections
Positive Control: Tumor cells in hemangiosarcomas
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