VEGF Antibody Staining Protocol for Immunohistochemistry

 

Description: VEGF (vascular endothelial growth factor) is a homodimeric, disulfide-linked glycoprotein involved in angiogenesis which promotes tumor progression and metastasis. It exhibits potent mitogenic and permeability inducing properties specific for the vascular endothelium. Of the four isoforms of VEGF, the smaller two, VEGF165 and VEGF121, are secreted proteins and act as diffusible agents, whereas the larger two (VEGF189 and VEGF206) remain cell associated.

 

Primary Antibody

Name: VEGF Antibody

Clone: Rabbit polyclonal

Supplier: Abcam

Catalog Number: ab46154

Dilution: 1:500 - 1:1000 depending on detection system 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

Counterstain
Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Results:
Staining Pattern: Cytoplasmic, cell surface, and extracellular matrix
Images: Search image

Additional Information:
Species Reactivity: Human, mouse, rat
Fixation: Formalin fixed paraffin sections
Positive Control: Skin (endothelial cells, sweet gland positive)
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: