Nis elements v 5

The Emx2 mouse riboprobe was a generous gift from T. Capellini and has been previously described^{33 (link)}. Whole-mount in situ hybridizations was performed using previously described protocols^{88 (link)}. In brief, mouse E11.5 and E13.5 (CD-1) embryos were post-fixed with 4% PFA in 1× PBS, washed with 1× PBS, treated with 20 μg ml⁻¹ proteinase in 1× PBT for 45 min and incubated overnight with the Emx2 riboprobe at 65 °C. The next morning, the probe was washed with MABT (Maleic acid, NaCl, Tween-20) and incubated overnight with secondary anti-DIG antibodies (1:2,000) diluted in MABT, 2% Boehringer Blocking Reagent and 20% heat-treated sheep serum. After washing several times with MABT, the signal was developed by incubating with NBT/BCIP. Once a signal had developed sufficiently, the reaction was stopped by washing several times with PBT and fixing in 4% PFA overnight. The embryos were visualized using a SMZ18 stereo microscope (Nikon). NIS Elements v5 (Nikon) was used to acquire microscopy images.

Immunofluorescence was performed on primary hepatocytes as previously described^{10 (link)}. Two different primary antibodies were used, Tomm20 (Cell Signaling, Cat#42406) and AIF (Cell Signaling, Cat#5318), and incubated overnight at 4°C. Alexa-Fluor 555-conjugated secondary antibodies (1:500, Thermo Fisher Scientific) were added for 1 h at room temperature. Slides were mounted in Mowiol 4-88 mounting medium (Sigma-Aldrich). Images were acquired using a filterless laser-scanning confocal microscope (Leica TCS SP5) using excitation wavelengths of 488 nm for green fluorescence (autofluorescence), 561 nm for red fluorescence (immunofluorescence), and 405 nm for DAPI (Sigma) nuclear labeling. Quantifications of Tomm20 and AIF fluorescence intensity per cell and per field of view were measured by processing raw data with NIS-Elements v.5.2 digital imaging analysis software (Nikon Instruments) for segmentation and precise quantification, implementing the general-analysis tool-box. AIF and Tomm20 mitochondrial fluorescence intensity levels were plotted as mean ± SEM values of each field of view. All the images were further processed with Photoshop CS6 (Adobe) software. For the statistical analysis of fluorescence intensity levels, the Mann–Whitney paired comparison was used.

Structured illumination microscopy (SIM) was performed on a Nikon N-SIM-S system (Ti-2 stand; Hamamatsu ORCA Flash 4.0 scientific CMOS dual cameras with Cairn splitter system; Nikon Perfect Focus; Chroma ET525/50m, ET595/50m and ET 700/75m emission filters; and Nikon laser bed with 405 nm/488 nm/561 nm/640 nm laser lines). A Nikon 100×1.49 NA TIRF oil objective was used. NIS Elements v5 software (Nikon) was used to control the system and acquire pre-expansion 3D-SIM images. Expanded samples were imaged on an ASI RAMM microscope frame. Widefield imaging was performed using a Nikon 100× TIRF (NA 1.45) objective and an Evolve Delta EM-CCD camera, via a quad-band emission filter (Semrock, 432/515/595/730 nm). iSPIM was performed using twin Nikon 40× (NA 0.8) water-dipping objectives, a similar quad-band emission filter (Semrock, 432/515/595/730 nm) and a Hamamatsu ORCA Flash 4.0 scientific CMOS camera. Illumination for both setups was from a Cairn Research laser bank containing 100 mW 405 nm, 150 mW 488 nm, 50 mW 561 nm and 100 mW OBIS 640 nm continuous wave (CW) lasers. Light was directed to the sample via a quad-band dichroic mirror (Semrock, 405/488/561/635 nm). Micro-Manager (https://micro-manager.org/) was used to control the system and scan the sample.

For mitochondrial and lysosome cell imaging, MNs plated in optical 4- or 8-well µ-Slides (Ibidi GmbH) pre-coated with poli-L-ornithine and laminin (both from Sigma Aldrich) were transduced with CellLight™ Mitochondria-RFP BacMam 2.0 and/or CellLight™ lysosome-GFP BacMam 2.0 reagents (both from ThermoFisher Scientific) (30 particles per cell) and incubated for 48 h at 37 °C. Imaging was performed with a Crest Optics Spinning Disk module (Crest-Crisel Instruments) mounted on a fully automated inverted Nikon ECLIPSE Ti microscope (Nikon) and acquisitions achieved with an Andor DU-888 EM-CCD camera (Andor) for fast recordings and NIS-Elements v.5 software (Nikon).