Expression of CD40 in human PMNs by Western blot analysis and localization of CD40 by digital microscopy. (A) Experiments were performed on 3 donors. Proteins from PMN whole cell lysates (1.25 × 106) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and were immunoblotted with a monoclonal antibody to human CD40. The CD40 glycoprotein band is present at 48 kDa. (B) Fixed PMNs were incubated with a human CD40 monoclonal antibody, and then the images were prepared for digital microscopy as previously described.12 (i) CD40 immunoreactivity is red because of the secondary donkey antimouse antibody conjugated to Cy3. (ii) The membrane is green because of the association of the wheat germ agglutinin (WGA)–AlexaFluor 488 with salicylic acids, and the nucleus is stained blue with the fluorescent bis-benzimide nuclear dye. (iii) Colocalization of CD40 and the plasma membrane is displayed in pseudocolor. Arrows point to the red area, where a high amount of colocalization is present. Blue represents low colocalization between CD40 and the plasma membrane. These images are representative of 3 different experiments on the PMNs of 3 donors.
