CD25 Antibody Staining Protocol for Immunohistochemistry


Description: CD25 antigen, the alpha subunit of interleukin-2 receptor, is a single-chain glycoprotein with a molecular weight of 55kD. Following the activation of T cells with antigen or mitogen in the presence of the monokine interleukin-1, interleukin 2 (IL-2) is rapidly synthesised and secreted. In response to this, a subpopulation of T cells expresses high affinity receptors for IL-2. These cells proliferate, expanding the T cell population which is capable of mediating helper, suppressor and cytotoxic functions. IL-2 receptor is not uniquely found on T cells and is expressed on HTLV-I transformed T and B cells, EBV transformed B cells, myeloid precursors and oligodendrocytes. It is absent on thymocytes, resting T cells, non-activated B cells and null cells.


Primary Antibody

Name: CD25 Antibody

Clone: Tu69, Mouse anti-Human

Supplier: Novocastra Labs

Catalog Number: NCL-CD25-305

Dilution: 1:100 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: 60min/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

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Membrane
Images: Search image

Additional Information:
Tissue Type: Tonsil, spleen, thymus
Fixation: Formalin fixed paraffin sections. Not recommended for acetone fixed frozen sections
Positive Control: Tonsil, spleen, thymus
Negative Control: Omit primary antibody, isotype control or 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