CD4 Antibody Staining Protocol for Immunohistochemistry

 

Description: CD4, a single chain transmembrane glycoprotein, is found on a T cell subset (helper/inducer) representing 45% of peripheral blood lymphocytes. It is also present on 80% of thymocytes and at a lower level on monocytes. It is involved in recognition of antigen presented along with MHC class II by APCs. It serves as receptor for HIV.

 

Primary Antibody

Name: CD4 Antibody

Clone: 1F6, Mouse monoclonal

Supplier: Lab Vision

Catalog Number: MS-392-S

Dilution: 1:10 - 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: 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). Other buffers (citrate, Tris-EDTA, etc.) has been tested but the results were NOT satisfactory.

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 - Recommended detection method.

Chromogen Substrate

Reagent: DAB

Incubation Time/Temperature: 1-3 minutes/room temperature

Counterstain

Reagent: Mayer's Hematoxylin

Staining Time: 30 seconds

Results:

Staining Pattern: Cell membrane

Images: Search image

Additional Information:

Species Reactivity: Human

Fixation: Formalin fixed, paraffin embedded sections

Positive Control: Human tonsil (T-cells)

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

 

References: