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CD34 Class II Antibody Staining Protocol for Immunohistochemistry
Description: CD34 is a single-chain transmembrane protein of approximately 116 kDa. CD34 is expressed on immature haematopoietic stem/progenitor cells, capillary endothelial cells, embryonic fibroblasts and rare glial cells in nervous tissue. CD34 appears to be expressed at its highest level on the earliest progenitors, and to decrease progressively with maturation. CD34 is a stage-specific, rather than a lineage-specific, leucocyte differentiation antigen. The most immature definable B-lymphoid precursors (CD19+/CD10+/TdT+) are CD34+. Immature T-lymphoid precursors also express TdT and CD34. Normal peripheral blood lymphocytes, monocytes, granulocytes, and platelets do not express CD34. Approximately 60% of acute Blymphoid leukaemias and 40% of acute myeloid leukaemias (AML), and 1% to 5% of acute T-lymphoid leukaemias express CD34. Chronic lymphoid leukaemias, lymphomas and multiple myelomas have been found to be uniformly CD34 negative. Monoclonal antibodies to CD34 can be confined to three main classes, class I, class II and class III, defined by the differential sensitivity of the corresponding CD34 epitopes to degradation by specific enzymes. The QBEnd 10 antibody is a class II monoclonal antibody that recognizes a CD34 epitope that is resistant to neuraminidase, and sensitive to glycoprotease and chymopapain.
Primary Antibody
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Name: CD34 Class II Antibody |
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Clone: QBEnd-10, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M7165 |
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Dilution: 1:50 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: Cytoplasmic of endothelial cells |
| Images: Search image |
Additional Information:
| Tissue Type: Skin, tonsil |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Capillaries of most tissues as well as umbilical artery and, to a lesser extent, vein |
| 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:
Not need to perform antigen retrieval on acetone fixed frozen sections |
References: