Whole-mount immunofluorescence shows highly fluorescent dots in zebrafish embryos injected with both wt and mutated hCRYAB and hMYOT. Whole-mount immunofluorescence analysis with specific myotilin and αb-crystallin antibodies was performed at 48 hpf, and images of muscle fibers in the trunk region were acquired with Axiozoom.V16 fluorescent stereomicroscope equipped with Axiocamera 506 (Zeiss International, Oberkochen, Germany). In (A,B), representative full Z-stack brightfield and fluorescent images are reported for zebrafish wildtype embryos not injected (a–a″ in A,B) or injected with hMYOT WT (b–b″ in A), hMYOT S95I (c–c″ in A), hCRYAB WT (b–b″ in B), hCRYAB G154S (c–c″ in B). Panels (a″, b″ and c″) both in (A,B) represent zoomed images from panels (a, b and c), respectively. Scale bar is 500 μm for (a,a′,b,b′,c,c′) panels and 100 μm for (a″,b″,c″) panels. Highly fluorescent dots of different shapes and sizes (white arrows) are indicated. (C). Bar graphs report the percentage of fish showing highly fluorescent dots (black closed bars). White open bars represent fish showing no fluorescent dots. Three independent experimental analyses have been performed. Total number of embryos analyzed for each experimental condition: wildtype not injected (n = 30 in (A) and left panel in (C); n = 35 in (B) and right panel in (C)), hMYOT WT (n = 30), hMYOT S95I (n = 56), hCRYAB WT (n = 20), hCRYAB G154S (n = 26).
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