Embryos were collected 2 h after being laid. They were dechorionated with 50% bleach for 2.75 min and mounted in Halocarbon oil 27 (Sigma) between a semipermeable membrane (Sarstedt) and coverslip (18 mm×18 mm). All movies were imaged at room temperature (∼23°C) with a Zeiss LSM800 confocal laser scanning microscope and Plan-Apochromat 40×/1.3 numerical aperture (N.A.) oil-immersion objective. 408 nm, 488 nm and 561 nm lasers were used to visualize His2Av-eBFP2, MCP-GFP and mCherry-PCP, respectively. To capture the full embryo, two adjacent tiles with 50 pixel overlaps were taken, resulting in a final image size of 950×500 pixels. A stack of 15 images with 0.7 µm step size in z were captured at each time point with a time resolution of 61 s/frame. For eve-MS2, seven biological replicates were taken for the wild-type embryos and five were taken for the Kr heterozygous embryos. Three replicates were taken for the wild-type and Kr heterozygous embryos for kni>MS2 and gt>MS2. The same laser setting was used between wild-type and Kr heterozygous embryos.
Semipermeable membrane
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A semipermeable membrane is a thin, selective barrier that allows the passage of certain molecules or ions while blocking others. It serves as a key component in various laboratory applications, facilitating the controlled transport of materials across the membrane.
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2 protocols using semipermeable membrane
1
Visualization of Drosophila Embryogenesis
2
Visualizing mRNA Dynamics in Drosophila Embryos
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Yw; nanos>SV40NLS-mCherry-PCP, His2Av-eBFP2/CyO; nanos>MCP-GFP virgins were crossed with males carrying the MS2/PP7 reporter genes.33 (link) Embryos from the cross were dechorionated and mounted in Halocarbon oil 27 (Sigma) between a semipermeable membrane (Sarstedt) and coverslip (18 mm × 18 mm). Embryos were imaged using a Zeiss LSM800 and Plan-Apochromat 40x/1.3 numerical aperture (N.A.) oil-immersion objective. The pixel size was set to 347 nm for the hb-MS2-lacZ-PP7 condition and 312 nm for all other images. A single image was 512 x 512 pixels. Images were created by taking a projection of the second-highest pixel value from a stack of 14 images separated by 0.75 μm, and the final time resolution was 29.13 s per frame. Typical images were started from the beginning of mitosis to about 25 min into NC14. Images were captured in 16-bit. Since some constructs exhibited too low transcriptional activity to be captured by the same laser power, we used several different laser powers that were optimized for each construct. Yet, the same laser power was used for all the constructs shown in Figure 2 , where we compared the mRNA production between different constructs. We confirmed that varying laser powers did not affect our measurement of the Pol II elongation.
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