Fas Antibody Staining Protocol for Immunohistochemistry


Description: Cytotoxic T lymphocyte (CTL)-mediated cytotoxicity constitutes an important component of specific effector mechanisms in immunosurveillance against virus-infected or -transformed cells. Two mechanisms appear to account for this activity, one of which is the perforin-based process. Independently, a FAS-based mechanism involves the transducing molecule FAS (APO-1) and its ligand (FAS-L). The human FAS (APO-1) protein is a 48 kDa cell surface glycoprotein that belongs to a family of receptors that includes CD40, nerve growth factor receptors and tumor necrosis factor receptors. The FAS antigen is expressed on a broad range of lymphoid cell lines, certain of which undergo apoptosis in response to treatment with antibody to FAS. These findings strongly imply that targeted cell death is potentially mediated by the intercellular interactions of FAS with its ligand or effectors, and may be critically involved in CTL-mediated cytotoxicity.


Primary Antibody

Name: Fas Antibody

Clone: B-10, Mouse anti-Human

Supplier: Santa Cruz Biotechnology

Catalog Number: sc-8009

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: 60 minutes/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 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: Testis, colon, spleen.
Fixation: Formalin fixed paraffin sections.
Positive Control: Testis, colon, spleen.
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


Not tested on frozen sections