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. 2015 May 11;126(4):520–530. doi: 10.1182/blood-2014-09-601484

Figure 2.

Figure 2

Tmod3−/− embryos show hemorrhages with abnormal platelets. (A) Representative images of whole Tmod3+/+ (left) and Tmod3−/− (right) embryos at E14.5. Bar, 1 mm. (B) Immunofluorescence staining for CD41 (gray) and Hoechst (blue) in cryosections of blood vessels of Tmod3+/+ (left) and Tmod3−/− (right) embryos, revealing circulating CD41+ platelets in situ. Bar, 10 µm. Images are single optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4), zoom 1. (C) Relative numbers of platelets in peripheral blood of Tmod3+/+ and Tmod3−/− E14.5 embryos. Tmod3+/+, 63 ± 20 (n = 4 fields from 2 Tmod3+/+ embryos); Tmod3−/−, 30 ± 7 (n = 5 fields from 2 Tmod3−/− embryos). *P < .05. (D) Representative images of Tmod3+/+ (top) and Tmod3−/− (bottom) platelets stained with CD41. Bar, 4 µm. Images are compressed Z stacks of optical sections acquired using a Zeiss LSM 780 confocal laser scanning fluorescence microscope with a ×100 oil-immersion objective (N.A. 1.4), zoom 3. (E) Platelet sizes in peripheral blood of Tmod3+/+ and Tmod3−/− embryos at E14.5, measured from images as in (B). Platelet diameters were determined from line scans across platelets using Volocity 6.3 software. Tmod3−/− average platelet diameters were ∼×1.5 greater than Tmod3+/+ platelets. Tmod3+/+, 1.73 ± 0.46 µm (n = 114); Tmod3−/−, 2.21 ± 0.76 µm (n = 98). ***P < .001. (F) Representative TEM images of Tmod3+/+ and Tmod3−/− platelets (left and middle panels; Bar, 1 µm), with high magnification views of circumferential microtubule rings (red arrows, right panel; Bar, 200 nm). Average WT platelet diameter in TEM images is ∼3 µm, similar to previous studies,10 but larger than measurements from fluorescence optical sections (B), which include measurements across the short and long axes of the asymmetric platelets (E).