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

Name: Active Caspase-3 Antibody

Clone: Rabbit anti-Active Caspase-3

Supplier: Millipore

Catalog Number: AB3623

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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