Figure 4

Expected results. (A) Resliced z-stacks along the x–z axis. 10 Embryos were composited together for each image. Embryos were either mounted in BABB (left) or Prolong Gold (right). (B) Signal intensity going through the mounted embryos—BABB (blue) and Prolong Gold (magenta). Solid colored line indicates the mean, lightly shaded regions are +/− 1 standard deviation. The black dotted line represents the hypothetical shape of an embryo without fluorescent decay. (C) Box plots comparing fluorescence intensity after reusing antibody solutions. Each point represents the average expression of an individual embryo (N = 10 embryos per condition). (D) Box plots comparing the amount of embryos lost during fixation using manual conditions (magenta, N = 10) or Flyspresso (blue, N = 19). Each point represents the percent loss from a single fixation. (E) Single representative embryos from 10 embryo pools using manual conditions and (F) the Flyspresso pipeline. (G) Box plots for the average nuclear intensity (left) and background intensities (right). Left: each point represents the average nuclear expression for a single embryo (N = 10 pools, n = 10 embryos per pool, 100 points total) between the manual pipeline (magenta) and the Flyspresso pipeline (blue). Right: the average intensity between the stripes for each represented embryo shown in panels e and f for the manual pipeline (magenta, N = 10) and the Flyspresso pipeline (blue, N = 10). (H) Boxplots for the normalized nuclear intensities, where each point is the average nuclear intensity for each pool for manual methods (magenta, N = 10 pools) and the Flyspresso pipeline (blue, N = 10 pools). (I and J) Reporter construct variant 173-2 for the E3N enhancer. (I) X-gal staining overview image for 173-2. Black asterisks (*) indicate embryos not at developmental stage 16 or not in the ventral orientation. (J) Antibody staining with Flyspresso overview image for 173-2. Green box indicates embryo clustering. (K) Adult Drosophila brain co-stained with a neuronal reporter and Elav. (l) 72hpf Zebrafish embryo stained with Flyspresso for Pax7 (left, magenta) and Myosin (right, green). For all box plots, the red line is the mean, light gray box is the Standard Error Mean (95% Confidence interval) and the whiskers +/− 1 standard deviation. p values are for a Student two-tailed t-test.