 |
|
CD68 Antibody Staining Protocol for Immunohistochemistry
Description: Macrosialin is a heavily glycosylated transmembrane protein of 87-115kD, which is specifically expressed by tissue macrophages, Langerhans cells and at low levels by dendritic cells. Macrosialin is the murine homologue of the human macrophage glycoprotein CD68, both are members of the lysosomal-associated membrane protein (lamp) family which are located predominantly within the cells and can be detected by flow cytometry using cell permeablisation. MCA1957 does detect surface macrosialin at low levels in resident mouse peritoneal macrophages which can be enhanced with thioglycollate stimulation.
Primary Antibody
|
Name: CD68 Antibody |
|
Clone: FA-11, Rat anti-Mouse |
|
Supplier: AbD Serotec |
|
Catalog Number: MCA1957 |
|
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. |
|
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) or IHC-TekTM Proteinase K Solution (Cat# IW-1101) |
| 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:
| Species Reactivity: Mouse. |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Bone marrow macrophages and mast cells, liver Kupffer's cells, lung macrophages, lymph node and tonsil germinal center macrophages |
| 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:
1. Not need to perform antigen retrieval for acetone fixed frozen sections. |
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