CD20 Antibody Staining Protocol for Immunohistochemistry


Description: This antibody can be used to identify B-cells in normal and neoplastic tissues. The L26 antibody reacts with two non-covalently bonded components weighing 33 kDa and 30 kDa present in the majority of B-cells. Positive staining with L26 occurs on the cytoplasmic side of the cell membrane. Positive staining was demonstrated using the L26 antibody and COS-1 cells transfected with cDNA encoding for the CD20 antigen. The L26 antibody detects an epitope which survives tissue processing, and is suitable for use on routinely fixed, paraffin-embedded tissues. The CD20 antigen is expressed in most B-cells present in peripheral blood and lymphoid tissue. The antigen is also found in most non-Hodgkin’s lymphomas of B-cell lineage. Cross-reactivity has not been observed with normal or malignant non-lymphoid cells except for: L26-positive dendritic cells in the medulla of the thymus and thymomas.


Primary Antibody

Name: CD20 Antibody

Clone: L26, Mouse anti-Human

Supplier: Dako

Catalog Number: M0755

Dilution: 1:300 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: Cytoplasmic side of the cell membrane
Images: Search image

Additional Information:
Tissue Type: Lymph node, tonsil, spleen, thymus
Fixation: Formalin fixed paraffin sections
Positive Control: Lymph node, 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