The cytosolic levels of free Ca2+ were measured using the fluorescent probe Fluo‐3 acetoxymethyl ester (Fluo‐3 AM; Invitrogen, Monza, Italy). Subconfluent SH‐SY5Y cells cultured on glass coverslips were incubated at 37°C for 5.0 min with 5.0 μM Fluo‐3 AM prior to exposure to D76N b2M aggregates for 1.0 hr. At the end of the incubation, the cells were fixed in 2.0% buffered paraformaldehyde for 10 min. Cell fluorescence was visualized using a confocal Leica TCS SP5 scanning microscope (Leica, Mannheim, Germany) equipped with a HeNe/Ar laser source for fluorescence measurements. The observations were performed using a Leica Plan 7 Apo X63 oil immersion objective, suited with optics for DIC acquisition. Cells from five independent experiments and three areas (about 20 cells/area) per experiment were analysed. The fluorescence intensity of Fluo3‐AM was analysed by using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and expressed as arbitrary units.
Plan 7 apo x63
Manufactured by Leica
The Plan 7 Apo X63 is a high-magnification, plan-apochromatic objective lens designed for use with Leica microscopes. It features a numerical aperture of 1.30 and a working distance of 0.17 mm, making it suitable for a variety of microscopy applications. The objective lens is optimized for superior optical performance and is capable of delivering high-resolution, high-contrast images.
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4 protocols using plan 7 apo x63
1
Measuring Cytosolic Ca2+ Levels
2
Amyloid-Beta Assay in N2a Cells
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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)).
3
Cytosolic Ca2+ Dynamics in Alzheimer's
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The cytosolic levels of free Ca2+ were measured using the fluorescent probe Fluo-3 acetoxymethyl ester (Fluo-3 AM; Invitrogen, Monza). Sub-confluent SH-SY5Y cells cultured on glass coverslips were incubated with 5.0 μM Fluo-3 AM at 37°C for 10 min prior to exposure to Aβ42 aggregates obtained after 72 h of aggregation in the absence or in the presence of each extract (100 μg/mL) for 30 min. At the end of the incubation, the cells were fixed in 2.0% buffered paraformaldehyde for 10 min. 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, suited with optics for DIC acquisition. Cells from five independent experiments and three areas (about 20 cells/area) per experiment were analyzed.
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] .
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