Subconfluent N2a cells grown on glass coverslips were treated for 24 h with Aβ42 samples (2.5 μM) grown in the presence or in the absence of AG and then washed with PBS. GM1 labeling was performed by incubating the cells with 10 ng/ml CTX-B Alexa 488 in complete medium for 10 min at room temperature. Then the cells were fixed in 2.0% buffered paraformaldehyde for 10 min and permeabilized by treatment with a 1:1 acetone/ethanol solution for 4.0 min at room temperature, washed with PBS, and blocked with PBS containing 0.5% BSA and 0.2% gelatin. After incubation for 1.0 h at room temperature with rabbit anti-Aβ42 polyclonal antibody diluted 1:600 in blocking solution, the cells were washed with PBS for 30 min under stirring and then incubated with Alexa 568-conjugated anti-rabbit secondary antibody (Molecular Probes) diluted 1:100 in PBS. Finally, the cells were washed twice in PBS and once in distilled water to remove non-specifically bound antibodies. Cell fluorescence was imaged using a confocal Leica TCS SP5 scanning microscope (Leica, Mannheim, Ge) equipped with a HeNe/Ar laser source for fluorescence measurements. The observations were performed using a Leica Plan 7 Apo X63 oil immersion objective (Nosi et al., 2012 (link)).
Tcs sp5 scanning microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 is a scanning microscope that provides high-resolution imaging capabilities. It is designed to capture detailed images of samples through the use of laser-based scanning technology. The TCS SP5 is a versatile instrument that can be used for a variety of applications in scientific research and analysis.
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Lab products found in correlation
Facscanto flow cytometer, by BD (5 mentions)
Plan 7 apo x63, by Leica (3 mentions)
Fluo 3 am, by Thermo Fisher Scientific (2 mentions)
H2dcfda, by Leica (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcf da, by Merck Group (1 mentions)
2 7 dichlorodihydrofluorescein diacetate dcfh2 da, by Merck Group (1 mentions)
Plan apo 63, by Leica (1 mentions)
Cyto id autophagy detection kit, by Enzo Life Sciences (1 mentions)
Plan apo, by Leica (1 mentions)
Fam flica caspase assay kit, by ImmunoChemistry Technologies (1 mentions)
8 isoprostane eia kit, by Cayman Chemical (1 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (1 mentions)
Plan apo 63x, by Leica (1 mentions)
Eclipse 80i microscope, by Nikon (1 mentions)
14 protocols using tcs sp5 scanning microscope
1
Amyloid-Beta Assay in N2a Cells
2
Visualizing ROS in Plant Leaves
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Trichogin GA IV-derived peptides at the concentration of 50 μM in water were applied as 10-μl droplets on the abaxial surface of detached Micro-Tom and A. thaliana leaves. Five droplets were applied on each leaf, and three leaves per treatment were analyzed. Incubations were performed in Petri dishes for 24 and 48 h as previously described. ROS production by leaves was visualized by staining with the cell-permeable probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Sigma-Aldrich, United States), as reported in Luti et al. (2020) (link). For staining, leaves were soaked for 1 h in a 10 μM H2DCFDA solution in 20 mM phosphate buffer, pH 6.8. After H2DCFDA incubation, leaves were washed twice with phosphate buffer and mounted on glass slides for microscopy analysis, which was performed under a confocal Leica TCS SP5 scanning microscope (Leica, Germany; λex 460 and λem 512 nm).
3
In situ H2O2 Visualization in A. thaliana
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A. thaliana leaves were treated with wtCP and mutants, and H 2 O 2 was visualized in situ according to Luti et al. [17] (link). Briefly, 10 μL drops of a 150 μM solution of each protein were applied on leaves and incubated for 24 h in a moist chamber at 22 °C. After incubation, droplets were removed and H 2 O 2 production was estimated by adding the specific probe 2′-7′ dichlorodihydrofluorescein diacetate (DCFH 2 -DA; SigmaAldrich, Saint Louis, MO, USA). Leaves were then incubated in a 20 mM sodium phosphate buffer at pH 6.8 containing 10 μM DCFH 2 -DA, at room temperature for 1 h. After staining, the samples were washed twice with fresh buffer to remove the excess of fluorophore and mounted in buffer on microscopic slides. Green fluorescence was then observed, using an excitation wavelength of 460 nm and emission wavelength at 512 nm, under a confocal Leica TCS SP5 scanning microscope (Leica, Mannheim, Germany).
4
GM1 Labeling and FRET Analysis of TTR Variants
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Subconfluent HL-1 cells grown on glass coverslips were treated for 24 h with the different samples of wt-TTR or L55P-TTR (20 μM) and then washed with PBS. GM1 labeling was performed by incubating the cells with 10 ng/ml CTX-B Alexa488 in complete medium for 10 min at room temperature. Then the cells were fixed in 2.0% buffered paraformaldehyde for 10 min and permeabilized by treatment with a 50% acetone/50% ethanol solution for 4.0 min at room temperature, washed with PBS and blocked with PBS containing 0.5% BSA and 0.2% gelatin. After incubation for 1.0 h at room temperature with a rabbit anti-TTR polyclonal antibody diluted 1:600 in the blocking solution, the cells were washed with PBS for 30 min under stirring and then incubated with Alexa568-conjugated anti-rabbit secondary antibody (Molecular Probes) diluted 1:100 in PBS. Finally, the cells were washed twice in PBS and once in redistilled water to remove non-specifically bound antibodies. Cell fluorescence was imaged using a confocal Leica TCS SP5 scanning microscope (Leica, Mannheim, Ge) equipped with a HeNe/Ar laser source for fluorescence measurements. The observations were performed using a Leica Plan 7 Apo X63 oil immersion objective. FRET analysis was performed by adopting the FRET sensitized emission method as previously reported [32] . 3D volume renderings were obtained by using the OsiriX software [32] .
5
Confocal Laser Scanning Microscopy Imaging
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CLSM was used for
imaging and analysis of cluster formations in the samples. CLSM snapshots
were monitored on a Leica TCS SP5 scanning microscope, operated in
the inverted mode, and equipped with a HeNe laser, λ = 543 nm.
Using two cover glasses, separated by a 120 μm spacer, and a
100×/1.4 NA oil immersion objective, imaging of samples was performed
by operating the microscope in a bright-field mode.
imaging and analysis of cluster formations in the samples. CLSM snapshots
were monitored on a Leica TCS SP5 scanning microscope, operated in
the inverted mode, and equipped with a HeNe laser, λ = 543 nm.
Using two cover glasses, separated by a 120 μm spacer, and a
100×/1.4 NA oil immersion objective, imaging of samples was performed
by operating the microscope in a bright-field mode.
6
Calcium Imaging of H3 Peptide Effects
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Aggregates of the H3 wt, H3 mutA and H3 mutE peptides (1 mM monomer concentration) were incubated in 50 mM sodium phosphate buffer, pH 7.2, 25°C under stirring, for 0 h, 15 days and 30 days. The samples were then diluted in cell culture media at a 100 μM peptide concentration, and then added to the SH-SY5Y cells seeded on glass coverslips for 60 min at 37°C. The cells were then loaded for 30 min at 37°C with 10 μM fluo3-AM (Life technologies, CA, USA), as previously described [27 ]. The resulting cell fluorescence was analysed by confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) equipped with an argon laser source for fluorescence measurements at 488 nm and a Leica Plan Apo 63× oil immersion objective. A series of optical sections (1024 × 1024 pixels) 1.0 μm in thickness was taken through the cell depth for each examined sample. The confocal microscope was set at optimal acquisition conditions, e.g. pinhole diameters, detector gain and laser powers. Settings were maintained constant for each analysis. To quantify the signal intensity of the fluorescent probe between 10 and 22 cells were analysed using ImageJ software (NIH, Bethesda, MD) and the fluorescence intensities expressed as arbitrary units.
7
Monitoring Autophagy Induction by Fluorescence Microscopy
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The Cyto-ID® Autophagy Detection Kit (Enzo Life Sciences) was used to monitor autophagy induction by fluorescence microscopy. The cells were plated for 24 h in 24-well plates containing coverslips and then treated with 10 μM natively folded or differently aggregated TTR-WT or TTR-L55P. Then, the medium was removed and the cells were washed twice with 100 μl of PBS and then incubated for 30 min at 37 °C in the dark with 100 μl of freshly diluted Cyto-ID® Green Detection Reagent (1:330). Finally, the cells were washed with Assay Buffer and analyzed by using a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany) equipped with an argon laser source for fluorescence measurements at 488 nm and a Leica Plan Apo 63 × oil immersion objective. A series of optical sections (1024 × 1024 pixels) 1.0 μm in thickness was taken through the cell depth for each examined sample. To quantify the green fluorescent signal, 10–22 cells were analyzed in each experiment using ImageJ software (NIH, Bethesda, MD).
8
Quantification of Mitochondrial Membrane Potential
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Mitochondrial membrane potential was assessed with tetramethylrhodamine, methyl ester, perchlorate (TMRM). TMRM is a lipophilic potentiometric dye that partitions between the mitochondria and cytosol in proportion to Δψ by virtue of its positive charge. At low concentrations, the fluorescence intensity is a simple function of dye concentration, which is in turn a direct function of mitochondrial potential. Therefore, the accumulation of dye in mitochondria and the intensity of the signal is a direct function of mitochondrial potential. For confocal microscope analysis, cells were cultured on glass cover slips and loaded with dye by adding the fluorescent probe TMRM, dissolved in 0.1% DMSO (100 nM final concentration), to the cell culture media for 20 min. at 37°C. The cells were fixed in 2.0% buffered paraformaldehyde for 10 min. at room temperature and the TMRM fluorescence analysed (at an excitation wavelength of 543 nm) with a confocal Leica TCS SP5 scanning microscope equipped with a helium-neon laser source for fluorescence measurements. Mitochondrial membrane potential was also quantified by flow cytometry. Single-cell suspensions were washed twice with PBS and incubated, in the dark, for 20 min. at 37°C with TMRM (100 nM) in DMEM. After labelling, cells were washed and resuspended in PBS and analysed with a FACSCanto flow cytometer (Becton-Dickinson).
9
Caspase Activation Assay in H9c2 Cells
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Since mitochondrial dysfunction is a well-known trigger of apoptosis, we next investigated the activation of pro-apoptotic initiator caspases 8 (extrinsic pathway) and 9 (intrinsic pathway), and effector caspase 3 and the possible influence of H + R, as described18 (link). Briefly, MnQ2 and MnM2. H9c2 cells seeded on glass coverslips were incubated with FAM-FLICA™ Caspase assay kit (Immunochemistry Technologies, Bloomington, MN, USA) for 30 min, following the manufacturer’s instructions. After incubation, the cells were thoroughly washed and fixed in 2% buffered paraformaldehyde for 10 min at room temperature. Fluorescence was detected by a confocal Leica TCS SP5 scanning microscope equipped with an argon laser source (excitation λ 488 nm) and a x63 oil immersion objective. Caspase activity was also quantified by flow cytometry, as previously reported18 (link),58 (link) single-cell suspensions were incubated with FAM-FLICA™ for 30 min at 37 °C, washed twice with PBS and analyzed using a FACSCanto flow cytometer (Becton-Dickinson).
10
Measuring Mitochondrial Membrane Potential
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Tetramethylrhodamine methyl ester perchlorate (TMRM) was used to assess the mitochondrial membrane potential. For confocal microscope analysis, cells were cultured on glass cover slips and loaded for 20 min at 37 °C with 100 nM TMRM (Life Technologies, Carlsbad, CA, USA). After fixing, cells were analyzed using a confocal Leica TCS SP5 scanning microscope (Mannheim, Germany). Mitochondrial membrane potential was also quantified by flow cytometry. Cells were incubated for 20 min at 37 °C with TMRM (100 nM) in DMEM and analyzed using a FACSCanto flow cytometer (Becton-Dickinson).
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