Dfc9000 scmos camera

Fluorescence microscopy was performed with a Thunder imaging system (Leica) equipped with a DFC9000 sCMOS camera, an HC PL Fluotar 20X air (N.A. 0.4) objective and using the LasX software (Leica).
Confocal microscopy was performed with a laser-scanning microscope (Leica DMi8) equipped with an ACS APO 63X oil DIC immersion objective. Images were acquired using the LasX software (Leica), with a resolution of 1024 × 1024 pixels and a number of Z-stacks (step size of 0.3 μm) encompassing the entire cell soma.

Images were acquired on a DMI6000 inverted fluorescent microscope (Leica) using an ORCA-ER B/W CCD camera (Hamamatsu; native resolution 1344 × 1024 pixels) through Metamorph software (Molecular Devices, version 7.6) or on a DMI8 inverted fluorescent microscope (Leica; native resolution 2048 × 2048 pixels) using a Leica DFC9000 sCMOS camera through LASX software (Leica). Axons and soma were imaged using ×10 and ×20 air lenses. Images of axons showing CM-H₂DCFDA, cyt c, and TMRE were imaged using ×63 oil lenses.

Skin, small intestine, and liver samples were collected from mice belonging to each experimental group, 3-weeks after transplant. Mouse skin biopsies were collected from the interscapular region of the mice. The small intestine samples were collected from the 2 cm region above the cecum, while the liver tissue was harvested from the left lobes. The samples were fixed for at least 48 h in 4% paraformaldehyde and embedded in paraffin. The sections were stained with hematoxylin and eosin (H & E fast staining kit, Carl Roth, Karlsruhe, Germany) according to manufacturer’s recommendation. The images were captured using a Leica DMi8 inverted microscope with a Leica DFC9000 sCMOS camera and processed using Leica LAS X software, version 3.3 (Leica Microsystems, Wetzlar, Germany).

After the heat shock treatment (50 °C), cells were stained with 5 µM H₂DCFDA (Sigma, D6883) for the detection of ROS accumulation, 5 µM C11-BODIPY™ 581/591 (Cayman, 27086) for the detection of lipid peroxidation, 3 µM JC-1 (Cayman, 10009172) for staining the mitochondrial membrane potential, 15 µm Calcium Green-1, AM (Cayman, 20400) for staining cytosolic calcium, and 1 µM FerroOrange (Merck, SCT210) for iron staining at room temperature for 20–30 min, followed by two washes with 1X PBS. Fluorescence images were captured using a Leica fluorescence microscope (Leica Microsystems, Germany) equipped with a Leica DFC9000 sCMOS camera. The excitation/emission of fluorescent dyes used the following filters: H₂DCFDA (Ex/Em: 492/515 nm), C11-BODIPY™ 581/591 (Ex/Em: 460–495/510–550 nm), JC-1 (Ex/Em: 488/538 nm for monomers and 596 nm for oligomers), Calcium Green-1 (Ex/Em: 506/531 nm), and FerroOrange (Ex/Em: 543/580 nm). For fluorescence intensity, 100 µL of stained cells were transferred into a black 96-well plate and fluorescence was measured using a Synergy HTX Multi-mode Plate Reader (BioTek, VT, USA) with the indicated excitation and emission wavelengths.