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Myeloperoxidase (MPO) Antibody Staining Protocol for Immunohistochemistry
Description: Myeloperoxidase (MPO), molecular weight 118 kDa, is a major constituent of cytoplasmic primary (azurophilic) granules of neutrophilic myeloid cells. The functional role of myeloperoxidase and other enzymes contained in primary granules is destruction of microorganisms and debris within the phagocytic vacuole of the cell. Since primary granules appear early in myeloid cell differentiation and are present throughout maturation, myeloperoxidase is a useful marker for for detection of myeloid cells.
Primary Antibody
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Name: Myeloperoxidase Antibody |
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Clone: Rabbit anti-Human polyclonal |
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Supplier: Dako |
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Catalog Number: A0398 |
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Dilution: 1:1000 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 (myeloid cells of both neutrophilic and eosinophilic types) |
| Images: Search image |
Additional Information:
| Tissue Type: Spleen |
| Fixation: Formalin fixed paraffin sections, acetone fixed frozen sections, or cell smears (no antigen retrieval needed for frozen sections or cell smears) |
| Positive Control: Spleen |
| 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 |
References: