Dfc360 camera

Fluorescent sections were imaged using Leica AF6000 epifluorescence microscope connected to a DFC360 camera on constant exposure settings for three channels, DAPI, Fgf8, and pErk, and analysed using IMAGEJ. IMAGEJ was used to draw 10-µm wide regions of interest (ROI) on each image (see Fig. S1 for illustration of ROI placement) and used to quantify signal intensity at increasing distances from the edge of the bead into the tissue (for Fgf8 and pErk gradient quantification) and in the outer edge of the bead (for Fgf8 only). Data were then combined and graphed using GraphPad Prism 6 (GraphPad). Images from all genotypes of each time point experiment were imaged, processed and analysed in parallel to minimise technical variation for comparison between genotypes of the same time point. Values from BSA control cultures were subtracted to remove background fluorescence. Fgf8 fluorescence in in vivo sections were quantified using IMAGEJ. Quantification of Fgf8 in vivo: mean fluorescence for boxes of 100×180 µm drawn at the CSB were obtained for at least 2 sections per embryo with N being number of embryos analysed. WT, N=4; Hs2st^−/−, N=3; Hs6st1^−/−, N=3.

On day 9 after transfection, culture medium was collected in 96-well plates and rat primary neuronal cultures were washed once with PBS. Cells were imaged in Live Cell Imaging Solution (LCIS, Thermo Fisher Scientific) containing 140 mM NaCL, 2.5 mM KCl, 1.8 mM CaCl₂, 1.0 mM MgCl₂, 20 mM HEPES, pH 7.4. Semi-automated image acquisition was performed using a fully automated Leica DMI 6000 microscope equipped with a Leica HCX PL FL L 20×/0.4 objective and a Leica DFC360 camera. The microscope was controlled by the Leica LAS AF v. 2.7 software with HCS A Matrix screener extension software. However, any automated research-grade inverted fluorescence microscope can be used for this type of assay, provided the controller software allows storing x-y coordinates to image the same fields of view (FOV) at subsequent time points. Using a 96-well template, 16 images per well from 10 wells per transfection were collected, adding up to a total of 160 images per condition. Likewise, on day 10 after transfection culture medium was removed, cells were washed once with 1× PBS and the same FOV were imaged 24 h after treatment. Images of transfected mouse cultures were used in Figure 2 and Supplementary Figures S1, S2, for illustration purposes.