Figure 3.
Platelet GPVI is a critical mediator of neutrophil recruitment and transmigration. Immunofluorescence staining of cryosections of the lungs of control or JAQ1-injected mice 4 hours after NaCl or LPS treatment. Images were acquired using a Thunder Imager DMi8 (Leica Microsystems) equipped with a 20× objective (HC PL APO 20×/0.80 DRY, Leica Microsystems) and a Leica-DFC9000GT-VSC12293 camera (Leica Microsystems). (A) Representative micrographs of whole left lung lobes showing extent of pulmonary neutrophil infiltration (Ly6G+, yellow). The dashed line depicts boundaries. Scale bar, 1 mm. (B) Magnified excerpts of a representative single FOV used to quantify neutrophils (Ly6G+, yellow) (left) and platelets (GPIX+, cyan) (right). Scale bar, 80 μm. (C-D) Quantification of neutrophil (C) (left graph) and platelet (D) numbers by manual counting. Neutrophils were identified as structures showing staining for 4′,6-diamidino-2-phenylindole (DAPI) (nucleus) and Ly6G. Spectrophotometric determination of MPO activity in whole lung lysates (C) (right graph). Each data point represents 1 mouse. (E) Representative confocal micrographs of neutrophils (Ly6G+, yellow), platelets (GPIX+, cyan), vessels (CD31+, magenta), and DAPI (nucleus, blue), exemplifying neutrophils counted as transmigrating (top) or intra-alveolar (bottom). Detailed representative images were acquired with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 63× objective (HC PL APO CS2 63×/1.40 OIL, Leica Microsystems). The dashed line marks the region of the magnified excerpt on the right. (Top) Scale bar, 10 μm; (bottom) scale bar, 30 μm. (F) Quantification of transmigrating neutrophils (left graph) by manual counting. Transmigrating neutrophils were identified as structures showing staining for DAPI (nucleus) and Ly6G, partially protruding into the AS. (G) Quantification of intra-alveolar neutrophils (left graph) by manual counting. Intra-alveolar neutrophils were identified as structures showing staining for DAPI (nucleus) and Ly6G, entirely inside the AS. Each data point represents the average number of neutrophils counted in 30 FOV for 1 mouse. Bars represent mean per group ± standard deviation. The two-way ANOVA with a Šidák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. AS, alveolar space.

Platelet GPVI is a critical mediator of neutrophil recruitment and transmigration. Immunofluorescence staining of cryosections of the lungs of control or JAQ1-injected mice 4 hours after NaCl or LPS treatment. Images were acquired using a Thunder Imager DMi8 (Leica Microsystems) equipped with a 20× objective (HC PL APO 20×/0.80 DRY, Leica Microsystems) and a Leica-DFC9000GT-VSC12293 camera (Leica Microsystems). (A) Representative micrographs of whole left lung lobes showing extent of pulmonary neutrophil infiltration (Ly6G+, yellow). The dashed line depicts boundaries. Scale bar, 1 mm. (B) Magnified excerpts of a representative single FOV used to quantify neutrophils (Ly6G+, yellow) (left) and platelets (GPIX+, cyan) (right). Scale bar, 80 μm. (C-D) Quantification of neutrophil (C) (left graph) and platelet (D) numbers by manual counting. Neutrophils were identified as structures showing staining for 4′,6-diamidino-2-phenylindole (DAPI) (nucleus) and Ly6G. Spectrophotometric determination of MPO activity in whole lung lysates (C) (right graph). Each data point represents 1 mouse. (E) Representative confocal micrographs of neutrophils (Ly6G+, yellow), platelets (GPIX+, cyan), vessels (CD31+, magenta), and DAPI (nucleus, blue), exemplifying neutrophils counted as transmigrating (top) or intra-alveolar (bottom). Detailed representative images were acquired with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 63× objective (HC PL APO CS2 63×/1.40 OIL, Leica Microsystems). The dashed line marks the region of the magnified excerpt on the right. (Top) Scale bar, 10 μm; (bottom) scale bar, 30 μm. (F) Quantification of transmigrating neutrophils (left graph) by manual counting. Transmigrating neutrophils were identified as structures showing staining for DAPI (nucleus) and Ly6G, partially protruding into the AS. (G) Quantification of intra-alveolar neutrophils (left graph) by manual counting. Intra-alveolar neutrophils were identified as structures showing staining for DAPI (nucleus) and Ly6G, entirely inside the AS. Each data point represents the average number of neutrophils counted in 30 FOV for 1 mouse. Bars represent mean per group ± standard deviation. The two-way ANOVA with a Šidák multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. AS, alveolar space.

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