Plan apochromat 63 1.4 oil dic objective

Fixed or live cells were imaged using a Zeiss LSM 5, Pascal Axiovert 200 confocal microscope (Carl Zeiss, Oberkochen, Germany), with a Plan-Apochromat ×63/1.4 Oil DIC objective and 488, 543 and 639 nm excitation lasers. In each independent experiment, 5–15 cells were evaluated and averaged [7 (link)]. For dynamic calcium measurements, images were acquired at 1 s intervals. Basal fluorescence was measured for 50 s, and then histamine-induced signals were imaged for 200 s. Data are expressed as fluorescence change relative to basal values ([F − F₀]/F₀). The area under the curve was quantified during the first 50 s post-stimulus. For static fluorescence measurements, images were deconvoluted, background-subtracted, thresholded and analysed using the ImageJ software. Colocalization analysis was performed within a single plane at the cell equator using the JACoP plugin. 3D object analysis was performed on cell reconstructions consisting of 10 z-planes using the 3D Object Counter plugin. ER cross-sectional area, mean mitochondrial area, mitochondrial DRP1 fluorescence and MTO/mtHSP70 fluorescence ratio per mitochondria were analysed within a single plane at the cell equator using the Analyze Particles function. For triple colocalization analysis, image intersections were obtained using the Image Calculator command of ImageJ (“AND” operator).

The HeLa S3 wild-type, GPC1 knockout, and GPC1 knockout + GPC1 cells used in Figures 2 and 4 were cultivated in µ-Slide 8 Well Glass Bottom dishes in the absence of doxycycline. This prevented the expression of FGF2-GFP so that endocytosis experiments with purified FGF2-GFP and fluorescent transferrin could be conducted without interference. For live cell imaging, cells were washed twice with cold Live Cell Imaging Solution (Thermo Fisher Scientific) and incubated for 5 min on ice. Cells were imaged with a Zeiss LSM 800 confocal microscope using a Zeiss Plan-APOCHROMAT 63×/ 1.4 Oil DIC objective. Imaging was started directly after replacing the solution with cold Live Cell Imaging Solution containing both 25 µg/ml Transferrin-Alexa Fluor 546 (Thermo Fisher Scientific) and 5 µg/ml recombinant FGF2-GFP (Steringer et al., 2017 (link)). Time-lapse videos were recorded with images being acquired every 10 s for a total of 20 min for each cell line. In parallel experiments, still images of fixed cells (4% PFA; Electron Microscopy Science) were taken at time points of up to 60 min using the same microscopy settings as indicated above. For time points 0, 5, and 10 min, different laser power and digital gain settings were used due to smaller fluorescent signals at shorter incubation times. Images and videos were processed using Fiji (Schindelin et al., 2012 (link)).