Figure 7
Figure 7. Localization of trapped neutrophils during diapedesis in cremaster venules by confocal fluorescence microscopy of stained tissue whole mounts. A 3D confocal image analysis of cremaster was used to determine sites of arrest of transmigrating leukocytes after the administration of blocking antibodies. Mice were intrascrotally stimulated with IL-1β and intravenously injected with anti-CD99 antibodies, anti-CD99L2 antibodies, or preimmune IgG as a control. Three hours later, whole mounts of cremaster muscles were immunostained for ESAM (green), laminin-α5 (red), and MRP-14 (blue) to visualize endothelial cell contacts, basement membrane, and neutrophils, respectively. Neutrophil localization relative to the endothelium and its underlying basement membrane was analyzed by confocal microscopy. (A) Fluorescent images showing a representative longitudinal vessel segment typically used for the 3D analysis and evaluation. The left panel shows an image of a venule stained for endothelial cell contacts (green) and neutrophils (blue) only. The image on the right depicts the same venule with the staining for the basement membrane (red) added. Projection images of only one-half of the venule have been used for better depiction. Bar represents 20 μm. (B) Schematic drawing illustrating the criteria used to define the positions of the neutrophils within the cremaster tissue samples. Position A indicates neutrophils embedded within the endothelial layer; position B, neutrophils located between endothelial cells and the basement membrane; and position C, fully emigrated neutrophils. (C) Representative images showing cross sections of venules with neutrophils located at the positions indicated in panel B (staining as in panel A). (D) The absolute numbers of neutrophils found at the positions indicated under the respective experimental conditions. Values are mean ± SD (on average, 20 of the most frequent vessel segments having 7-16 neutrophils in total analyzed). (E) Numbers of neutrophils found at the positions indicated expressed as percentage of total neutrophils. Here, all vessel segments having more than 5 neutrophils in total have been evaluated (on average, 30 vessel segments). co indicates control. ***P < .001. **P < .01. Statistical analysis was done by ANOVA evaluating all available data.

Localization of trapped neutrophils during diapedesis in cremaster venules by confocal fluorescence microscopy of stained tissue whole mounts. A 3D confocal image analysis of cremaster was used to determine sites of arrest of transmigrating leukocytes after the administration of blocking antibodies. Mice were intrascrotally stimulated with IL-1β and intravenously injected with anti-CD99 antibodies, anti-CD99L2 antibodies, or preimmune IgG as a control. Three hours later, whole mounts of cremaster muscles were immunostained for ESAM (green), laminin-α5 (red), and MRP-14 (blue) to visualize endothelial cell contacts, basement membrane, and neutrophils, respectively. Neutrophil localization relative to the endothelium and its underlying basement membrane was analyzed by confocal microscopy. (A) Fluorescent images showing a representative longitudinal vessel segment typically used for the 3D analysis and evaluation. The left panel shows an image of a venule stained for endothelial cell contacts (green) and neutrophils (blue) only. The image on the right depicts the same venule with the staining for the basement membrane (red) added. Projection images of only one-half of the venule have been used for better depiction. Bar represents 20 μm. (B) Schematic drawing illustrating the criteria used to define the positions of the neutrophils within the cremaster tissue samples. Position A indicates neutrophils embedded within the endothelial layer; position B, neutrophils located between endothelial cells and the basement membrane; and position C, fully emigrated neutrophils. (C) Representative images showing cross sections of venules with neutrophils located at the positions indicated in panel B (staining as in panel A). (D) The absolute numbers of neutrophils found at the positions indicated under the respective experimental conditions. Values are mean ± SD (on average, 20 of the most frequent vessel segments having 7-16 neutrophils in total analyzed). (E) Numbers of neutrophils found at the positions indicated expressed as percentage of total neutrophils. Here, all vessel segments having more than 5 neutrophils in total have been evaluated (on average, 30 vessel segments). co indicates control. ***P < .001. **P < .01. Statistical analysis was done by ANOVA evaluating all available data.

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