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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
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Name: CD34 Antibody |
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Clone: My10, Mouse anti-Human |
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Supplier: Becton Dickinson |
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Catalog Number: 347660 |
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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. |
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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 |
Counterstain
| Reagent: Mayer's Hematoxylin |
| Staining Time: 30 seconds |
Results:
| 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 |
Notes:
CD34 (Clone QBEnd10, DakoCytomation, Cat# M7156) also works well at 1:100 dilution with Tris-EDTA pH 9.0 pretreatment |
References: