Cleaved Caspase-3 Antibody Staining Protocol for Immunohistochemistry


Description: Caspase-3 (CPP-32, Apoptain, Yama, SCA-1) is one of the key executioners of apoptosis, as it is either partially or totally responsible for the proteolytic cleavage of many key proteins such as the nuclear enzyme poly (ADP-ribose) polymerase (PARP). Activation of caspase-3 requires proteolytic processing of its inactive zymogen into activated p17 and p12 fragments. Cleavage of caspase-3 requires aspartic acid at the P1 position. Cleaved Caspase-3 (Asp175) (5A1) Rabbit Monoclonal Antibody detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. This antibody does not recognize full length caspase-3 or other cleaved caspases. 


Primary Antibody

Name: Cleaved Caspase-3 Antibody

Clone: Rabbit anti-Cleaved Caspase-3

Supplier: Cell Signaling Technology

Catalog Number: 9661

Dilution: 1:100 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: Cytoplasmic/nuclear
Images: Search image

Additional Information:
Tissue Type: Mouse embryo, pancreas
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Mouse embryo, pancreas
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


There is no need to perform antigen retrieval for acetone fixed frozen sections