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CD68 Antibody Staining Protocol for Immunohistochemistry
Description:
CD68 is a 110 kD transmembrane glycoprotein which is
highly expressed in human monocytes including tissue macrophages and
was identified to be a member of a group of hematopoietic molecules
including CD43, CD34 and the lymph node ligand for L-selectin
GlyCAM-1.1 The antigen is thought to play a role in endocytosis
and/or lysosomal traffic.
Primary Antibody
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Name: CD68, Macrophage Antibody |
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Clone: KP11, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M0814 |
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Dilution: 1:2000 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 |
| Images: Search image |
Additional Information:
| Tissue Type: Skin |
| Fixation: Formalin fixed paraffin sections |
| Positive Control: All tissue macrophages such as kidney, pancreas, skin, etc. |
| 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:
1. Kunisch
E., et al (2003) Macrophage specificity of three anti-CD68
monoclonal antibodies (KP1, EBM11, and PGM1) widely used for
immunohistochemistry and flow cytometry.
Ann Rheum Dis.
63(7):774-84.
2. Caffo M., et al (2005) CD68 and CR3/43 immunohistochemical expression in secretory meningiomas. Neurosurgery. 57(3):551-7; discussion 551-7. PubMed Abstract
3. Li WV, et al (1996) Immunohistochemical characterization of mast cell disease in paraffin sections using tryptase, CD68, myeloperoxidase, lysozyme, and CD20 antibodies. Mod Pathol. 9(10):982-8. PubMed Abstract