A Nikon-Microphot-SA microscope was used to capture images of DAB-stained embryos (40×). Images for the Sxl RNAi rescue experiment were acquired using a 100×, 1.49 NA Apo TIRF oil immersion objective on a Nikon Ti-E system fitted with a Yokagawa CSU-X1 spinning disk head, Hamamatsu Orca Flash 4.0 v2 digital CMOS camera, and Nikon LU-N4 solid state laser launch. Imaging for all other smFISH and fluorescent immunostaining experiments was performed on a Nikon A1 inverted laser-scanning confocal microscope.
Images were assembled using ImageJ (NIH) and Adobe Photoshop and Illustrator software to crop regions of interest, adjust brightness and contrast, generate maximum-intensity projections, and separate or merge channels. To assess the spreading of the RNAs or protein in different mutant backgrounds compared to the control we generated plot profiles using ImageJ. The posterior-most 75 µm of each embryo was plotted for comparison, and embryos from a single biological replicate are plotted in figures given that variation between fluorescence between replicates obscured the pole plasm distribution trends if embryos from all replicates were plotted together.
Images were assembled using ImageJ (NIH) and Adobe Photoshop and Illustrator software to crop regions of interest, adjust brightness and contrast, generate maximum-intensity projections, and separate or merge channels. To assess the spreading of the RNAs or protein in different mutant backgrounds compared to the control we generated plot profiles using ImageJ. The posterior-most 75 µm of each embryo was plotted for comparison, and embryos from a single biological replicate are plotted in figures given that variation between fluorescence between replicates obscured the pole plasm distribution trends if embryos from all replicates were plotted together.