Factor VIII Antibody Staining Protocol for Immunohistochemistry


Description: Factor VIII is a glycoprotein present in human plasma. Factor VIII related antigen has been reported present in human endothelial cells. It is also present in megakaryocytes and platelets. Immunohistochemical staining for factor VIII related antigen could be used to determine if the benign and malignant neoplastic lesions are of endothelial origin. Since factor VIII related antigen is present in normal plasma and platelets, positive staining of intravascular plasma inevitably occurs in areas of hemorrhage or areas where vascular damage and leakage of plasma are present. Therefore, specific staining must be differentiated from non-specific background.


Primary Antibody

Name: Factor VIII Related Antigen Antibody

Clone: Rabbit polyclonal

Supplier: Zymed Labs

Catalog Number: 18-0018

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: Incubator
Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101)
Heat/Cool Temperature: 37 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 (endothelial cell)
Images: Search image

Additional Information:
Tissue Type: Tonsil, liver, kidney.
Fixation: Formalin fixed paraffin sections
Positive Control: Tonsil, liver, kidney.
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