CD45 Antibody Staining Protocol for Immunohistochemistry

 

Description: This antibody reacts with both alloantigens and all isoforms of the CD45 leukocyte common antigen (LCA), also known as Ly-5 or T200, found on all cells of hematopoietic origin, except erythrocytes. CD45 is a transmembrane glycoprotein which is expressed at high levels on the cell surface, and its presence distinguishes leukocytes from non-hematopoietic cells. The CD45 isoforms play complex roles in T-cell and B-cell antigen receptor signal transduction. This antibody is specific for mouse CD45 and suitable for immunohistochemical staining of Zinc-fixed and formalin fixed paraffin sections as well as acetone-fixed, frozen sections.

 

Primary Antibody

Name: CD45 Antibody

Clone: 30-F11, Rat anti-Mouse

Supplier: BD Pharmingen

Catalog Number: 550539

Dilution: 1:20 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

Counterstain
Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Results:
Staining Pattern: Membrane
Images: Search image

Additional Information:
Species Reactivity: Mouse
Fixation: Formalin fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Thymus, spleen
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

Notes:

Not need to perform antigen retrieval on acetone fixed frozen sections

 

References: