CD8 Antibody Staining Protocol for Immunohistochemistry


Description: The CD8 molecule is composed of two chains and has a molecular weight of 32kD. It is found on a T cell subset of normal cytotoxic/suppressor cells which make up approximately 20 to 35 percent of human peripheral blood lymphocytes. The CD8 antigen is reported to be detected on natural killer cells, 80 percent of thymocytes, on a subpopulation of 30 percent of peripheral blood null cells and 15 to 30 percent of bone marrow cells.


Primary Antibody

Name: CD8, T Cell Antibody

Clone: 4B11, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-CD8-4B11

Dilution: 1:20 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: Membrane of T Cells
Images: Search image

Additional Information:
Tissue Type: Tonsil
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Tonsil, lymph node, thymus
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 need to perform antigen retrieval for acetone fixed frozen sections