Af6000 epifluorescence microscope

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.

Fluorescent images were captured with a Leica AF6000 epifluorescence microscope connected to a DFC360 camera while bright-field images were captured using a Leica DLMB microscope coupled to a DFC480 camera. Exposure settings were kept constant throughout to enable comparisons between samples. Figures were edited using Adobe Illustrator and Photoshop CS6.

For mitochondrial potential measurements, the cells were plated in individual Petri dishes with a glass bottom FluoroDish cell culture dish, 35 mm (WPI FD35-100, Sarasota, FL, USA). Tetramethylrhodamine (TMRE; 100 nM; Thermo Fisher T669, Waltham, MA, USA) was added to the culture media and incubated for 30 min at 37 °C, 5% CO₂. Hereafter, the cells were washed three times with Krebs buffer and maintained in this medium at room temperature for the rest of the experiment. A time-lapse series (200 pictures in 12 min) was made using a Leica AF-6000 epifluorescence microscope. After 1 min of base line measurements, rotenone (4 µM) (Sigma R8875, St. Louis, MO, USA) and antimycin-A (2 µM) (Sigma A8674, St. Louis, MO, USA) were dissolved in Krebs buffer and added to the medium. One minute before the end of the time lapse, FCCP (20 µM) (Enzo BML-CM120-0010, Farmingdale, NY, USA) was added. Imaging analysis was performed using ImageJ (NIH, Bethesda, MD, USA).

Fluorescence-labeled sections were imaged using either a Leica AF6000 epifluorescence microscope coupled to a Leica DFC360 digital camera or a Nikon Ti: E Inverted confocal microscope. DAB stained and in situ hybridized sections were imaged using a Leica DLMB microscope coupled to a Leica DFC480 color digital camera.

For mitochondrial potential measurements, hPSC-CMs plated in individual petridishes with glass bottoms (35 mm) were used 1 day after the medium change. Tetramethylrhodamine (TMRE; 1200 nM; T669, ThermoFisher (Waltham, MA, USA)) and Mitotracker Green (MTG; 100 nM; M7514; ThermoFisher (Waltham, MA, USA)) were added to the culture media and incubated for 30 min at 37 °C, 5% CO₂. Hereafter, the hPSC-CMs were washed 3 times with CDM3. A time-lapse (200 pictures in 12 min) was made using with a Leica AF-6000 epifluorescence microscope (Leica (Wetzlar, Germany)). After 1 min, Rotenone (4 µM) and Antomycin-A (2 µM) were added to the CDM3 medium. One min before the end of the time-lapse, (20 µM) were added. Imaging analyses were performed using ImageJ.
For Mitochondrial calcium release, hPSC-CMs plated in individual Petri dishes with glass bottoms (35 mm) were used 1 day after the medium change. Fluo4-AM (5 µM; F14201; ThermoFisher (Waltham, MA, USA)) and Thapsigargine (10 µM; BML-PE180-001; Enzo life sciences (Bruxelles, Belgium) were added to the culture media and incubated for 230 min at 37 °C, 5% CO₂. Hereafter, the hPSC-CMs were washed 3 times with Ca²⁺-free Krebs buffer. A time-lapse (200 pictures in 30 min) was made using with a Leica AF-6000 microscope. After 2 min, FCCP (20 µM) suspended in Krebs buffer was added. Imaging analyses were performed using ImageJ.

The JC‐1 assay kit (Beyotime) was employed to evaluate the alteration in the mitochondrial membrane potential (MMP, △Ψm) by measuring the change in the JC‐1 levels. JC‐1 selectively enters mitochondria, exhibiting different fluorescence properties in accordance with changes in MMP. With elevated MMP, JC‐1 polymerizes and exhibits a crimson luminescence. While with lower MMP, it exists as a monomer and fluoresces green. The cells were subjected to QDN concentrations for a period of 48 hours. Cells were then examined and visualized with an inverted Leica AF6000 epifluorescence microscope (Leica).