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GFP Antibody Staining Protocol for Immunohistochemistry
Description: A highly versatile antibody that gives a stronger signal than other anti-GFP antibodies available. On Western blot the antibody detects the GFP fraction from cell extracts expressing recombinant GFP fusion proteins and has also been shown to be useful on mouse sections fixed with formalin. In immunocytochemistry, the antibody gives a very good signal on recombinant YES-GFP chimeras expressed in COS cells. It is routinely used in immunoprecipitation (IP) and IP-Western protocols and has been used successfully in immunohistochemistry on formalin fixed paraffin embedded tissue sections. This antibody is reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP and EGFP.
Primary Antibody
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Name: GFP Antibody |
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Clone: Rabbit polyclonal |
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Supplier: Novus Biologicals |
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Catalog Number: NB600-303 |
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Dilution: 1:2,000 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 minutes/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 ºC - 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 |
| Images: Search image |
Additional Information:
| Species Reactivity: Reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP and EGFP |
| Fixation: Formalin fixed paraffin sections, paraformaldehyde or acetone fixed frozen sectoins, formaldehyde or methanol fixed cultured cells. |
| Positive Control: GFP expressing tissues or cells. |
| Negative Control: Omit primary antibody, isotype control or 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 |
Notes:
No need to perform antigen retrieval on frozen sections or cultured cells. |
References: