Bone marrow plasma cells are closely associated with megakaryocytes. (A) Cryosections of murine bone marrow (femur) after secondary immunization with Ova. Sections were stained for Ig κ light chain (top left, red), fluorochrome-coupled Ova (top right, blue), and the megakaryocyte marker CD41 (bottom left, green). Overlay of the 3 colors (bottom right) reveals contact between an Ova-specific plasma cell (Ig κ⁺/Ova binding, red and blue merges to pink) and a large CD41⁺ megakaryocyte. Analysis was performed with a Leica TCS-SL confocal microscope and processed with Leica software (LCS Lite Version 2.61 Build 1538) and Adobe Photoshop CS3. Approximately 30% of the Ova-specific plasma cells were found to colocalize with megakaryocytes during the whole period of observation (6-150 days after secondary immunization; in total, ∼ 500 Ova-specific plasma cells from 15 mice were analyzed using a 60×/0.135 NA oil objective). (B) Plasma cells and megakaryocytes are frequently found in close contact with each other. Ig κ light chain was used as a marker for plasma cells (“Methods”). Femurs of unimmunized mice were stained for Ig κ light chain (red) and the megakaryocyte marker CD41 (green); nuclei are stained with Sytox-orange (blue). Arrows indicate plasma cells in contact with polyploid CD41⁺ megakaryocytes (top left). Larger magnification images are shown in the top right and bottom panels (40×/0.95 NA oil objective). (C) Staining controls. (Top panels) Specificity of Ova staining was tested by blocking with unlabeled Ova. (Left section) Bone marrow sections were stained for Ig κ (green) and Ova (red). (A 40×/0.95 NA oil objective was used.) (Right section) No Ova-positive cells were detectable anymore after blockade with unlabeled protein. For that purpose, sections were preincubated with 100 excess unlabeled Ova and subsequently stained for Ig κ (green) and Ova (red). (Bottom panels) Bone marrow section stained for (left) nuclei (blue) and CD41 (green) or (right) rat IgG-isotype control.