Figure 2
Figure 2. In vivo endothelial dome formation in WT mice visualized by whole-mount staining of the cremaster muscle and 2-photon microscopy. The cremaster of C57BL/6 WT mice was fixed in paraformaldehyde and stained for platelet endothelial cell adhesion molecule-1 (PECAM-1; green, Alexa 488) and MRP-14 (red, Alexa 568) 3 hours after application of IL-1β into the scrotum. (A) Overview merge of a postcapillary venule showing a migrating neutrophil covered by a dome (arrows in box; scale bar: 10 μm). (B-D) Magnifications of the area in panel A showing the single channels for PECAM-1 (B), MRP-14 (C), and the merge (D). The dome is highlighted by arrows. Presumably rolling and adherent cells not covered by a dome are highlighted by open arrows. Scale bar represents 5 μm. Image acquisition, panels A-D: Zeiss LSM510Meta confocal fluorescent microscope on Axiovert 200M (Zeiss), 40×/1.2 NA water C-Apochromat (Zeiss); DAKO fluorescent mounting medium; LSM510Meta photo-multiplier tubes (Zeiss); LSM image Examiner Version 4.0.0.241 (Zeiss). (E) Percentage quantification of the whole-mount staining based on the number of cells interacting with the vasculature/vessel in the field of view in WT vs Lsp1−/− animals. Eleven to 16 vessels per whole mount and per background (n = 5) were analyzed. ***P < .001. Error bars indicate SEM. For 2-photon microscopy, the cremaster of lys-EGFP mice was superfused with 5nM KC and the endothelium stained with anti–PECAM-1 Ab coupled to Alexa 594. (F) PECAM-1 positive stained endothelium demonstrating the formation of a dome (arrow). (G) Neutrophils (green) migrate and are encapsulated by endothelial domes (red) as highlighted by arrows. Insets represent magnifications of the dome and the encapsulated neutrophil from panels G and F. The dome reaches into the vessel lumen (L) and is highlighted by the arrow. Image acquisition, panels F-G: Olympus FV300 laser scanning confocal unit on Olympus BX61WI; Olympus 20×/0.95 NA water XLUMPLAN FI; bicarbonate superfusion buffer (132mM NaCI, 4.7mM KCI, 1.2mM MgSO4, Olympus Fluoview (FV300 O3D V5.0). (H) Percentage quantification of the 2-photon microscopy images in WT vs Lsp1−/− animals. Calculations were based on the number of cells interacting with the vasculature/vessel in the field of view (5- 8 vessels per mouse were observed; each group contained at least 5 animals). ***P < .001. Error bars indicate SEM. Scale bar represents 50 μm.

In vivo endothelial dome formation in WT mice visualized by whole-mount staining of the cremaster muscle and 2-photon microscopy. The cremaster of C57BL/6 WT mice was fixed in paraformaldehyde and stained for platelet endothelial cell adhesion molecule-1 (PECAM-1; green, Alexa 488) and MRP-14 (red, Alexa 568) 3 hours after application of IL-1β into the scrotum. (A) Overview merge of a postcapillary venule showing a migrating neutrophil covered by a dome (arrows in box; scale bar: 10 μm). (B-D) Magnifications of the area in panel A showing the single channels for PECAM-1 (B), MRP-14 (C), and the merge (D). The dome is highlighted by arrows. Presumably rolling and adherent cells not covered by a dome are highlighted by open arrows. Scale bar represents 5 μm. Image acquisition, panels A-D: Zeiss LSM510Meta confocal fluorescent microscope on Axiovert 200M (Zeiss), 40×/1.2 NA water C-Apochromat (Zeiss); DAKO fluorescent mounting medium; LSM510Meta photo-multiplier tubes (Zeiss); LSM image Examiner Version 4.0.0.241 (Zeiss). (E) Percentage quantification of the whole-mount staining based on the number of cells interacting with the vasculature/vessel in the field of view in WT vs Lsp1−/− animals. Eleven to 16 vessels per whole mount and per background (n = 5) were analyzed. ***P < .001. Error bars indicate SEM. For 2-photon microscopy, the cremaster of lys-EGFP mice was superfused with 5nM KC and the endothelium stained with anti–PECAM-1 Ab coupled to Alexa 594. (F) PECAM-1 positive stained endothelium demonstrating the formation of a dome (arrow). (G) Neutrophils (green) migrate and are encapsulated by endothelial domes (red) as highlighted by arrows. Insets represent magnifications of the dome and the encapsulated neutrophil from panels G and F. The dome reaches into the vessel lumen (L) and is highlighted by the arrow. Image acquisition, panels F-G: Olympus FV300 laser scanning confocal unit on Olympus BX61WI; Olympus 20×/0.95 NA water XLUMPLAN FI; bicarbonate superfusion buffer (132mM NaCI, 4.7mM KCI, 1.2mM MgSO4, Olympus Fluoview (FV300 O3D V5.0). (H) Percentage quantification of the 2-photon microscopy images in WT vs Lsp1−/− animals. Calculations were based on the number of cells interacting with the vasculature/vessel in the field of view (5- 8 vessels per mouse were observed; each group contained at least 5 animals). ***P < .001. Error bars indicate SEM. Scale bar represents 50 μm.

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