Caspase-9 Antibody Staining Protocol for Immunohistochemistry


Description: Caspase-9 is a member of the caspase family of cysteine proteases that have been implicated in apoptosis and cytokine processing. Caspases have been shown to be activated during normal human keratinocyte differentiation and this activation is required for thecnormal loss of the nucleus. In the presence of ATP, apoptotic stimuli induce proteolytic processing and activation of downstream caspases such as caspase-3, 6 and 7 initiating the caspase cascade. Caspase-9 is essential for apoptosis during the normal development of the central nervous system.


Primary Antibody

Name: Caspase-9 Antibody

Clone: 2C9B11, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-CASP-9

Dilution: 1:20 - 1:40 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
Images: Search image

Additional Information:
Tissue Type: Inflamed large bowel
Fixation: Formalin fixed paraffin sections
Positive Control: Inflamed large bowel
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