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CD45.1 Antibody Staining Protocol for Immunohistochemistry
Description: The A20 antibody reacts with CD45 (Leukocyte Common Antigen) on all leukocytes of mouse strains expressing the CD45.1 alloantigen (eg, RIII, SJL/J, STS/A, DA). This alloantigen was originally named Ly-5.2, and this was the designation at the time that the antibody was characterized. The designation was later changed from Ly-5.2 to Ly-5.1 to conform with the convention that the alloantigen designations be assigned to the C57BL/6 strain. mAb A20 does not react with leukocytes of most other mouse strains, which express the CD45.2 alloantigen. CD45 is a member of the Protein Tyrosine Phosphatase (PTP) family: Its intracellular (COOH-terminal) region contains two PTP catalytic domains, and the extracellular region is highly variable due to alternative splicing of exons (designated A, B, and C, respectively), plus differing levels of glycosylation. The CD45 isoforms detected in the mouse are cell type-, maturation-, and activation state-specific. The CD45 isoforms play complex roles in Tcell and B-cell antigen receptor signal transduction. The A20 antibody has been reported to inhibit some responses of B cells, from mice expressing the CD45.1 alloantigen, to antigens5 and LPS
Primary Antibody
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Name: CD45.1 Antibody, Biotinylated |
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Clone: A20, Mouse anti-CD45.1 |
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Supplier: BD Pharmingen |
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Catalog Number: 553774 |
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Dilution: 1:200 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: Antigen retrieval is not needed |
| Buffer/pH value: N/A |
| Heat/Cool Temperature: N/A |
| Heat/Cool Time: N/A |
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/cytoplasmic |
| Images: Search image |
Additional Information:
| Tissue Type: Spleen, thymus |
| Fixation: Acetone fixed frozen sections |
| Positive Control: Spleen, thymus |
| 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: