Figure 4.
Figure 4. Immune histochemical analysis of normal bone marrow for CD. Serial sections from B5-fixed bone marrow biopsies from healthy donors were subjected to antigen retrieval and incubated with a monoclonal antibody against CD68 (A). The arrow shows macrophages with long cellular processes staining positive for CD68, whereas the arrowhead shows a smaller positively stained mononuclear cell with no cellular processes (similar to the cell shown in panel F). Panel B shows staining with rabbit polyclonal antibody against CXCL7. The arrow points to a positively stained megakaryocyte, also identifiable by large size and multiple nuclei. The arrowhead shows a smaller mononuclear cell positive for CXCL7. The bound antibodies were detected with the HRP-conjugated Envison Plus system (DakoCytomation). Sections stained with appropriate isotype control antibodies are shown in panel C (monoclonal mouse IgG3 antibody) and panel D (polyclonal rabbit antibody). Panels E and F show double immune histochemical staining for CXCL7 and CD68 in bone marrow biopsies from healthy donors. CD68 (blue) is visualized with the Vectastain ABC-AP kit and Vector Blue, and CXCL7 (red) by EnVision Plus/HRP and NovaRED substrate. As shown in panel E, distinct patterns of staining for both CD68 and CXCL7 are observed as in panels A and B, but a small proportion of mononuclear cells without extensive cellular processes also stain for both of the antigens, suggesting that a subset of bone marrow monocytes also express CXCL7. No counterstain was used for panels E and F. All images were acquired using a Nikon Eclipse E800 microscope (Nikon Instruments, Kanagawa, Japan), Coolsnap CF Color Camera (Photometrics, Tucson, AZ), and Metamorph software (Molecular Devices, Sunnyvale, CA). For panels A-D and F, a 100 ×/1.3 NA objective with oil immersion was used; for panel E, a 60 ×/1.4 NA objective with oil immersion was used. Images were adjusted for brightness and contrast with Adobe Photoshop 7.0 (Adobe, San Jose, CA). Panel F was magnified 3 times after acquisition for better cellular detail.

Immune histochemical analysis of normal bone marrow for CD. Serial sections from B5-fixed bone marrow biopsies from healthy donors were subjected to antigen retrieval and incubated with a monoclonal antibody against CD68 (A). The arrow shows macrophages with long cellular processes staining positive for CD68, whereas the arrowhead shows a smaller positively stained mononuclear cell with no cellular processes (similar to the cell shown in panel F). Panel B shows staining with rabbit polyclonal antibody against CXCL7. The arrow points to a positively stained megakaryocyte, also identifiable by large size and multiple nuclei. The arrowhead shows a smaller mononuclear cell positive for CXCL7. The bound antibodies were detected with the HRP-conjugated Envison Plus system (DakoCytomation). Sections stained with appropriate isotype control antibodies are shown in panel C (monoclonal mouse IgG3 antibody) and panel D (polyclonal rabbit antibody). Panels E and F show double immune histochemical staining for CXCL7 and CD68 in bone marrow biopsies from healthy donors. CD68 (blue) is visualized with the Vectastain ABC-AP kit and Vector Blue, and CXCL7 (red) by EnVision Plus/HRP and NovaRED substrate. As shown in panel E, distinct patterns of staining for both CD68 and CXCL7 are observed as in panels A and B, but a small proportion of mononuclear cells without extensive cellular processes also stain for both of the antigens, suggesting that a subset of bone marrow monocytes also express CXCL7. No counterstain was used for panels E and F. All images were acquired using a Nikon Eclipse E800 microscope (Nikon Instruments, Kanagawa, Japan), Coolsnap CF Color Camera (Photometrics, Tucson, AZ), and Metamorph software (Molecular Devices, Sunnyvale, CA). For panels A-D and F, a 100 ×/1.3 NA objective with oil immersion was used; for panel E, a 60 ×/1.4 NA objective with oil immersion was used. Images were adjusted for brightness and contrast with Adobe Photoshop 7.0 (Adobe, San Jose, CA). Panel F was magnified 3 times after acquisition for better cellular detail.

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