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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
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Name: VEGF Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Abcam |
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Catalog Number: ab46154 |
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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. |
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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: 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: