Apoptosis Protease Activating Factor 1 Antibody Staining Protocol for Immunohistochemistry


Description: Apoptosis is one of a number of phenotypic responses that may occur as a result of signal transduction pathways occurring in the cell. One identified mechanism for initiating caspase activation requires the participation of mitochondria and involves apoptosis protease activating factor-1 (Apaf-1). Apaf-1 is a cytosolic protein that rests in a latent state until bound by cytochrome c (Apaf-2). Cytochrome c is commonly released from the mitochondria during apoptosis induced by many but probably not all cell death stimuli. The resulting Apaf1/cytochrome c complex associates with the zyymogen form of caspase-9 (Apaf-3) in the presence of dATP or ATP, promoting the autocatalytic activation of caspase-9. Once activated caspase-9 can then cleave and activate procaspase-3 directly, resulting in a cascade of additional caspase activation and apoptosis.


Primary Antibody

Name: Apoptosis Protease Activating Factor 1 Antibody

Clone: Rabbit polyclonal

Supplier: Novocastra Labs

Catalog Number: NCL-APAF1

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: Myocardium or skin, testis
Fixation: Formalin-fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Myocardium or skin, testis
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


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