CD40 Antibody Staining Protocol for Immunohistochemistry


Description: The 3/23 antibody reacts with CD40, a 40-50–kDa glycoprotein expressed on B lymphocytes, follicular dendritic cells, bone marrow-derived dendritic cells, thymic epithelium, and interdigitating cells in the T-cell zones of secondary lymphoid organs. The CD40 molecule has a central role in B cell growth and differentiation. Ligation of CD40 is reported to be an important signal in B-cell survival. Costimulation with anti-CD40 and IL-4 rescues B cells from apoptosis induced by extensive cross-linking of surface IgM or IgD. Furthermore, signaling through CD40 in combination with IL-4 reportedly induces immunoglobulin isotype switching and secretion of IgE by human B cells. Ligation of CD40 with the 3/23 antibody can induce splenic B cells to express the costimulatory molecule CD86 (B7-2).


Primary Antibody

Name: CD40 Antibody

Clone: Rat anti-Mouse

Supplier: Pharmingen

Catalog Number: 550285

Dilution: 1:50 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: Incubator
Buffer/pH value: IHC-TekTM Proteinase K Solution (Cat# IW-1101)
Heat/Cool Temperature: 37 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: Spleen, thymus
Fixation: Zinc fixative fixed paraffin sections, or acetone fixed frozen sections
Positive Control: Spleen, thymus, B cells, dendritic cells
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


Not need to perform antigen retrieval on acetone fixed frozen sections