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CD45 (Leucocyte Common Antigen) Antibody Staining Protocol for Immunohistochemistry
Description: CD45 is a transmembrane glycoprotein expressed on most nucleated cells of haematopoietic origin. CD45, encoded by a single gene mapped to chromosome 1, has various isoforms based on differential splicing of exons 4, 5 and 6. On human leucocytes, five different isoforms of CD45, named ABC, AB, BC, B and 0, have been identified. These isoforms are recognized by CD45RA, CD45RB, CD45RC and CD45R0 antibodies. The isoforms range in Mr from 180 000 to 220 000. All the CD45 isoforms share the same intracellular segment, which has been shown to have tyrosine phosphatase activity. Various leucocytes express characteristic CD45 isoforms, thus T cells express CD45 isoforms corresponding to their development and activation, B cells predominantly express the ABC isoform, and monocytes and dendritic cells predominantly express the B and 0 isoforms. Granulocytes principally express only the B and 0 isoforms.
Primary Antibody
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Name: CD45, Leucocyte Common Antigen Antibody |
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Clone: 2B11 + PD7/26, Mouse anti-Human |
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Supplier: Dako |
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Catalog Number: M0701 |
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Dilution: 1:400 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:
| Species Reactivity: Human |
| Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections |
| Positive Control: Tonsil, lymph node, 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 |
Notes:
Not need to perform antigen retrieval on acetone fixed frozen sections |
References: