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Bcl-2 Oncoprotein Antibody Staining Protocol for Immunohistochemistry
Description: Bcl-2 is a member of a family of proteins that are involved in apoptosis. Bcl-2 is an integral inner mitochondrial membrane protein of 25kD and has a wide tissue distribution. It is considered to act as an inhibitor of apoptosis. For this reason, bcl-2 expression is inhibited in germinal centres where apoptosis forms part of the B cell production pathway.
Primary Antibody
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Name: Bcl-2 Oncoprotein Antibody |
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Clone: bcl-2/100/D5, Mouse Anti-Human |
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Supplier: Novocastra Labs |
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Catalog Number: NCL-bcl-2 |
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Dilution: 1:50 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: 60min/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: Membrane and cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Tonsil |
| Fixation: Formalin-fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Tonsil |
| 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 |
Notes:
There is no need to perform antigen retrieval on acetone fixed frozen sections |
References: