Human Tissue Factor (CD142) Antibody Staining Protocol for Immunohistochemistry


Description: Two monoclonal antibodies resulting from the immunization of mice with purified apoprotein of human tissue factor (Mr = 47,000 D). The hybridomas secreting these antibodies were generated by the fusion of Balb/c spleen cells with the P3Ag8.653.1 myeloma cell line. The antibodies are purified from cell culture (No. 4508) or mouse ascites fluid (No. 4509) via Protein G affinity chromatography. The monoclonals react with and neutralize the purified apoprotein of human tissue factor, native human brain and placental thromboplastin, recognize tissue factor on the surface of tumor cells and LPS stimulated monocytes. At very high concentrations, Product No. 4509 will inhibit rabbit TF.

Primary Antibody

Name: Mouse Anti-Human Tissue Factor (CD142) Antibody

Clone: Mouse monoclonal antibody

Supplier: American Diagnostica Inc.

Catalog Number: 4508 and 4509

Dilution: 1:100 - 200 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 or overnight at 4 C.

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: Membrane/Cytoplasmic of endothelial cells.
Images: Search image

Additional Information:
Species Reactivity: Human
Tissue Type: Bran, placenta, cardiac tissue, pancreatic tissue, carotid and atherosclerotic arteries.
Fixation: Formalin fixed paraffin sections or acetone fixed frozen sections
Positive Control: Human Malaria tissues (brain, cardiac). Will not be positive on normal tissue.
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

Notes: Antigen retrieval is not needed for frozen sections.