Caspase-7 Antibody Staining Protocol for Immunohistochemistry

 

Description: Caspase-7 (CMH-1, Mch3, ICE-LAP3) has been identified as a major contributor to the execution of apoptosis. Caspase-7 is an effector caspase (along with caspase-2 and -3), meaning that it cleaves essential cellular machinery rather than activating other caspases. Caspase-7 is cleaved by many enzymes, including caspases-3, -6, -8, -9 and granzyme B. Once activated, caspase-7 cleaves many of the same substrates as caspase-3, including poly (ADP-ribose) polymerase, or PARP.

 

Primary Antibody

Name: Cleaved Caspase-7 (Asp198) Antibody

Clone: Polyclonal Rabbit ant-Human

Supplier: Cell Signaling Technology

Catalog Number: 9491

Dilution: 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: 60 min/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 ºC - 100 ºC/room temperature

Heat/Cool Time: 20 minutes/20 minuters

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: Cytoplasmic/membrane

Images: Search image

Additional Information:

Species Reactivity: Human, mouse, rat

Fixation: Formalin fixed paraffin sections.

Positive Control: Spleen

Negative Control: Omit primary antibody, isotype control, 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: