CD4 Antibody Staining Protocol for Immunohistochemistry


Description: The CD4 molecule (T4) is a single chain transmembrane glycoprotein with a molecular weight of 59kD. The CD4 antigen is found on a T cell subset (helper/inducer) representing 45 per cent of peripheral blood lymphocytes. It is also present on 80 per cent of thymocytes and at a lower level on monocytes.


Primary Antibody

Name: CD4 Antibody

Clone: 1F6, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-CD4-1F6

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 (T cells)
Images: Search image

Additional Information:
Tissue Type: Tonsil
Fixation: Formalin fixed paraffin sections
Positive Control: Tonsil, spleen, 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