Caspase-8 Antibody Staining Protocol for Immunohistochemistry


Description: The caspases represent a family of sulphydryl proteases which play important regulatory roles within the cell. Caspase-8, also called FLICE, has an N-terminal domain with sequence homology to the death effector domain of FADD that allows association of caspase-8 with the TNF/Fas family of receptors. This association with the cell surface death receptors has shown caspase-8 to be a proximal regulator of apoptosis. Caspase-8 is activated by association with the Fas/FADD death-inducing signaling complex to release two active subunits, p18 and p10, into the cytosol where they activate other caspases amplifying the apoptotic signal.


Primary Antibody

Name: Caspase-8 Antibody

Clone: 11B6, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-CASP-8

Dilution: 1:30 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: Testis
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Testis
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