CD34 Antibody Staining Protocol for Immunohistochemistry


Description: The CD34 antigen is a single-chain trans-membrane glycoprotein, Mr 105 to 120 kilodaltons (kd). The antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leucocyte antigen. Clone My10 and clone 8G12 recognize two distinct CD34 epitopes; at least three epitopes have been identified.The CD34 antigen is present on immature hematopoietic precursor cells and all hematopoietic colony-forming cells in bone marrow and blood, including unipotent (CFU-GM, BFU-E) and pluripotent progenitors (CFU-GEMM, CFU-Mix, and CFU-Blast). Terminal deoxynucleotidyl transferase-positive B- and T-lymphoid precursors in normal bone marrow are CD34+. The CD34 antigen is present on early myeloid cells that express the CD33 antigen but lack the CD14 and CD15 antigens and on early erythroid cells that express the CD71 antigen and dimly express the CD45 antigen. The CD34 antigen is also found on capillary endothelial cells and approximately 1% of human thymocytes. Normal peripheral blood lymphocytes, monocytes, granulocytes, and platelets do not express the CD34 antigen. CD34 antigen density is highest on early hematopoietic progenitor cells and decreases as cells mature. The antigen is absent on fully differentiated hematopoietic cells. Uncommitted CD34+ progenitor cells are CD38- and lack lineage-specific antigens such as CD71, CD33, CD10, and CD5, while CD34+ cells that are lineage-committed express the CD38 antigen in high density. Most CD34+ cells reciprocally express either the CD45RO or CD45RA antigens, with the CD45RO+ population being the more primitive. Approximately 60% of acute B-lymphoid leukemias and acute myeloid leukemias (AMLs) and 1% to 5% of acute T-lymphoid leukemias express the CD34 antigen. The antigen is not expressed on chronic lymphoid leukemias or lymphomas.

Primary Antibody

Name: CD34 Antibody

Clone: My10, Mouse anti-Human

Supplier: Becton Dickinson

Catalog Number: 347660

Dilution: 1:80 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 min/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
Images: Search image

Additional Information:
Tissue Type: Bone marrow, thymus
Fixation: Formalin fixed paraffin sections
Positive Control: Bone marrow, thymus
Negative Control: Omit primary antibody, isotype control, 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


CD34 (Clone QBEnd10, DakoCytomation, Cat# M7156) also works well at 1:100 dilution with Tris-EDTA pH 9.0 pretreatment