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

Name: CD45.1 Antibody, Biotinylated

Clone: A20, Mouse anti-CD45.1

Supplier: BD Pharmingen

Catalog Number: 553774

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.

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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

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