4 dpf
Et(Gal4-VP16)zc1044A;Tg(UAS-1b:nsfB-mCherry)c264larvae were treated with MTZ (10 mM, 5 h) and washed in 12-well plates as
described above. Larvae were then anesthetized with tricaine, placed in the
middle of each well of a 24-well flat bottom plate, and mounted with a drop
of low melting agarose under a stereomicroscope. The wells were infected
with 500 μL E3 containing 5 × 106/mL
FTR1-GFP-R. arrhizus spores. Imaging was performed at
28°C in the IncuCyte ZOOM HD/2CLR time lapse microscopy system
(Sartorius) equipped with an IncuCyte ZOOM 10 x PLAN FLUOR objective
(Sartorius). Phase contrast, red (800 ms) and green (400 ms) fluorescence
channels were imaged every 3 hours. 36 frames were captured per well. The
Basic Analyzer image analysis tool was used to track and quantify fungal
growth in the larval tissue. The algorithm was optimized using a training
image collection as previously described (Wurster et al., 2019 (link)). The GFP-positive area was quantified both
absolutely (in μm2) and in relation to the red fluorescent
area (%). To create time lapse movies, stacks of images were exported in
tagged image file format(tif) using the Time Plot function in the IncuCyte
ZOOM software. Videos were assembled and annotated in Microsoft PowerPoint,
and exported in MP4 format.
Et(Gal4-VP16)zc1044A;Tg(UAS-1b:nsfB-mCherry)c264larvae were treated with MTZ (10 mM, 5 h) and washed in 12-well plates as
described above. Larvae were then anesthetized with tricaine, placed in the
middle of each well of a 24-well flat bottom plate, and mounted with a drop
of low melting agarose under a stereomicroscope. The wells were infected
with 500 μL E3 containing 5 × 106/mL
FTR1-GFP-R. arrhizus spores. Imaging was performed at
28°C in the IncuCyte ZOOM HD/2CLR time lapse microscopy system
(Sartorius) equipped with an IncuCyte ZOOM 10 x PLAN FLUOR objective
(Sartorius). Phase contrast, red (800 ms) and green (400 ms) fluorescence
channels were imaged every 3 hours. 36 frames were captured per well. The
Basic Analyzer image analysis tool was used to track and quantify fungal
growth in the larval tissue. The algorithm was optimized using a training
image collection as previously described (Wurster et al., 2019 (link)). The GFP-positive area was quantified both
absolutely (in μm2) and in relation to the red fluorescent
area (%). To create time lapse movies, stacks of images were exported in
tagged image file format(tif) using the Time Plot function in the IncuCyte
ZOOM software. Videos were assembled and annotated in Microsoft PowerPoint,
and exported in MP4 format.