Procedures were carried out as previously reported [35 (link),36 (link)] and in accordance with Stellaris fluorescence in situ hybridization (FISH) instructions (www.biocat.com (accessed on 20 July 2022)). Through an online probe design tool (www.biosearchtech.com/stellarisdesigner/ (accessed on 10 August 2022), specific probes were chosen from input sequences (PARTICLE NR_038942.1; GAS5 NR_152521.1) for optimal binding properties to the target RNA. Probe fluorophores 5′carboxyfluorescein FAM (excitation (Ex): 495 nm; emission (Em): 520 nm) were chosen for the detection of these lncRNAs. Confocal fluorescence microscopic imaging was acquired with a Leica TCS SP8X confocal system (Leica Microsystems, Mannheim, Germany) using an HCX PL APO 40_/1.30 Oil objective and appropriate excitation using 488 nm and emission using 509 nm lasers. Emitted fluorescence signals were sampled at a resolution of 30 nm/pixel with a dwell time of 1.5 μs. Image analysis was carried out as indicated above.
Tcs sp8 x confocal system
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 X is a confocal system designed for advanced microscopy applications. It features a flexible platform, enabling the integration of multiple laser sources and detectors to accommodate a wide range of sample types and imaging requirements. The system's core function is to provide high-resolution, three-dimensional imaging capabilities through the use of confocal laser scanning technology.
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5 protocols using tcs sp8 x confocal system
1
Quantitative Imaging of lncRNA Expression
2
Oleogel Microstructural Visualization
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A 0.2% aqueous solution of Fast Green and Nile Red (Sigma Aldrich, Milan, Italy) was used to stain, respectively, the proteins and the oil of the oleogel samples. After staining, the samples were gently mixed by hand, placed on the microscope slide, covered with a cover slide and observed using a confocal laser scanning microscope at 100× magnification (Leica TCS SP8 X confocal system, Leica Microsystems, Wetzlar, Germany). Images were imported in jpeg format using the software LasX 3.5.5 (Leica Microsystems, Wetzlar, Germany).
3
Confocal Microscopy Analysis of Lipid Droplets
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A 0.2% aqueous solution of Fast Green and Nile Red was used to stain, respectively, the proteins and the oil. The hangingdrop method was used 20 to analyze the samples collected after the gastric and intestinal digestion phases. After staining, agarose (1%, w/w) was added ti the samples in a sample : agarose ratio of 1 : 2 (v/v). An amount of 2 µL of the obtained mix was placed on a microscope cover-slip and left to set for 1 min. The cover-slip with the gelled droplet was then fixed on a concave microscope slide and observed using a confocal laser scanning microscope at 100× magnification (Leica TCS SP8 X confocal system, Leica Microsystems, Wetzlar, Germany). Images were imported in the jpeg format using the software LasX 3.5.5 (Leica Microsystems, Wetzlar, Germany). Oil droplet dimension was determined based on the image analysis of confocal micrographs using Image-Pro Plus 6.3 (Media Cybernetics Inc., USA). Images were converted to 8-bit grey scale and software calibration was applied. Droplet diameter data were provided by the software and further elaborated to obtain the D 32 (Microsoft® Excel®).
4
Quantifying Lipid Droplet Dynamics in Hepatocytes
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HepG2 and Hepa1-6 cells were plated on collagen-coated 8-well μ-slide glass bottom slides (Ibidi, Martinsried, Germany). The next day, cells were transduced with AV-Hilpda in complete DMEM at 5 × 106 IFU/mL media and left overnight. HepG2 cells were incubated with a mixture of oleate and palmitate (ratio 2:1, total concentration 0.8 mM) for 8 h to promote LD formation. Hepa 1–6 cells were incubated with 1 mM of oleate:palmitate for 24 h. Cells were washed with PBS, fixed for 15 min with 3.7% formaldehyde, stained with 3 μg/mL of BODIPY® 493/503 and Hoechst for 45 min, and mounted with VECTASHIELD-H (Vector Laboratories). Cells were imaged on a Leica confocal TCS SP8 X system equipped with a 63 × 1.20 NA water-immersion objective lens. BODIPY® 493/503 was excited at 488 nm, and fluorescence emission was detected using internal Hybrid (HyD) in a spectral window of 505–578 nm. Images were acquired at 1,024 × 1,024 pixels, with pinhole set at 1 AU. Images were processed and analyzed with Fiji. Briefly, images were converted to binary, watershed, and LD size and number was measured with particle analysis set 0.07 μm2-infinity.
5
Lipid Droplet Imaging in Hepatocytes
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HepG2 and Hepa1-6 cells were plated on collagen-coated 8-well µ-slide glass bottoms (ibidi, Martinsried, Germany). The next day, cells were transduced with AV-Hilpda in complete DMEM at 5x10 6 IFU/mL media and left overnight. HepG2 cells were incubated with a mixture of oleate and palmitate (ratio 2:1, total concentration 0.8 mM) for 8h to promote LD formation. Hepa 1-6 cells were incubated with 1 mM oleate:palmitate for 24h. Cells were washed with PBS, fixed for 15 min with 3.7% formaldehyde, stained with 3 µg/mL BODIPY® 493/503 and Hoechst for 45 min, and mounted with Vectashield-H (Vector Laboratories). Cells were imaged on a Leica confocal TCS SP8 X system equipped with a 63× 1.20 NA water-immersion objective lens. BODIPY® 493/503 was excited at 488 nm and fluorescence emission was detected using internal Hybrid (HyD) in a spectral window of 505nm -578nm. Images were acquired 1024 × 1024 pixels with pinhole set at 1 AU, pixel saturation was avoided. Images were processed and analyse with Fiji. Briefly, images were converted to binary, watershed and LD size and number was measured with particle analysis set 0.07 µm 2 -infinity.
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