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Active Caspase-3 Antibody Staining Protocol for Immunohistochemistry
Description: Caspase-3 (CPP32, Apopain) is the most extensively studied apoptotic protein. Caspase-3 is synthesized as an inactive proenzyme (32 kDa) that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another upstream protease. The processed form of caspase-3 consists of large (17 kDa) and small (12 kDa) subunits which associate to form an active enzyme. The active caspase-3 proteolytically cleaves and activates other caspases, as well as relevant targets in the cells such as PARP and DFF.
Primary Antibody
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Name: Active Caspase-3 Antibody |
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Clone: Rabbit anti-Active Caspase-3 |
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Supplier: Millipore |
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Catalog Number: AB3623 |
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Dilution: 1:20 - 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: Gill's Hematoxylin or Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| Staining Pattern: Perinuclear |
| Images: Search image |
Additional Information:
| Species Activity: Human, Mouse, Rat |
| Tissue Type: Tonsil |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: Tonsil and appendix tissue. Jurkat cells induced for apoptosis with recombinant soluble TRAIL and staurosporine treated mouse 3T3 cell lysates |
| 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 |
References: