Localization of CD40 mutants in the ER. Intracellular double staining of CD40 (AlexaFluor568) with either calnexin, an ER marker (AlexaFluor488), or giantin, a Golgi Apparatus marker (AlexaFluor488-conjugate), in BLCLs from control (C) and from P2 and P5 patients. A further double staining was performed on C either with TGN46, a Trans Golgi Network marker (AlexaFluor488), or after cell incubation with fluorescently labeled transferrin (AlexaFluor488-conjugate) that localizes to the recycling endosomal compartment. Colocalization is visible as yellow staining in the merged micrographs. Scale bars, 5 μm. Analysis was performed by the LSM510META confocal microscope (Zeiss) with a EC Plan-Neofluar 100×/1.30 oil objective and a scan zoom of 1-1.5 ×. Digital images were analyzed with the LSM Image Browser (Zeiss) and processed with Adobe Photoshop 6.0 (Adobe Systems).