HeLa cells were transfected with the indicated plasmids by FuGENE. After transfection for 20 h, the cells were fixed with 4% paraformaldehyde for 15–20 min and then washed with PBS for 3 times. The nuclei were stained with DAPI for 2 min and then washed with PBS for 3 times. Imaging of the cells was carried out using Nikon A1 MP confocal microscope under a ×60 oil objective.
A1 mp confocal microscope
Manufactured by Nikon
The Nikon A1 MP confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a confocal optical design that allows for the capture of high-resolution, three-dimensional images of samples. The A1 MP provides users with a versatile platform for a wide range of research and diagnostic needs.
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5 protocols using a1 mp confocal microscope
1
Visualizing Transfected HeLa Cells
2
Confocal Imaging of Mitochondrial Membrane Potential
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Confocal imaging was performed with a Nikon A1–MP confocal microscope. First, 1 µL of JC–9 1 mM stock solution (Molecular Probes Inc., Eugene, OR, USA) was added per milliliter of culture medium. For the evaluation of mitochondrial membrane potential, images were acquired in two separated channels (excitation: 488 nm, emission: 525/50 nm for the green channel and 595/50 nm for the red channel). The red/green fluorescent ratio, which reflects variations in mitochondrial membrane potential, was calculated as:
where and are the fluorescence emission intensities in the red and green channels, respectively [36 (link),37 (link)]. The gain from the detectors and the laser intensity were kept fixed in all the experiments. Mitochondrial depolarization, indicated by a blue shift, corresponds to a decrease in the red/green fluorescent ratio, while a hyperpolarization results in an increased ratio [38 (link)].
where and are the fluorescence emission intensities in the red and green channels, respectively [36 (link),37 (link)]. The gain from the detectors and the laser intensity were kept fixed in all the experiments. Mitochondrial depolarization, indicated by a blue shift, corresponds to a decrease in the red/green fluorescent ratio, while a hyperpolarization results in an increased ratio [38 (link)].
3
Proximity Ligation Assay for HSV-1 Infection
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The PLA reagents were purchased from Sigma and the experiments were performed following the manufacturer’s instructions. Briefly, MEFs were seeded on Teflon-coated glasses and cultured for 18 h. After HSV-1 infection or transfection of HSV120 for 4 h, cells were fixed with 4% paraformaldehyde for 15–20 min at room temperature followed by permealization with 0.1% TritonX-100 for 10 min. Cells were blocked in 5% BSA in PBS for 30 min and incubated in primary antibodies for 2 h. Cells were washed with wash buffer A (DUO82049) for 3 times and incubated with the secondary antibodies with PLA probes (DUO92004-30RXN, DUO92002-30RXN) for 2 h at 37 °C followed by 3 times wash with the wash buffer A. The cells were then incubated with the Ligation-Ligase solution (DUO92008) for 30 min at 37 °C followed by 3 times wash with the wash buffer A. Cells were incubated with Amplification-Polymerase solution (DUO92008) for 100 min at 37 °C followed by 3 times wash with the wash buffer B (DUO82049). The nuclei were stained with DAPI for 2 min and then washed with PBS for 3 times. Imaging of the cells was carried out using Nikon A1 MP confocal microscope under a ×60 oil objective.
4
Th17 Differentiation and Retinal Immunostaining
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Naïve CD4+ T cells were isolated and cultured under Th17 differentiation condition for 3 days, washed, and fixed with 4% PFA at RT for 10 min. Cells were then permeabilized in PBS containing 0.2% Triton X-100 and 1% bovine serum albumin (BSA) for 10 min at RT, blocked by 5% BSA in PBS for 30 min and incubated with indicated primary antibodies overnight at 4 °C. After incubation with secondary antibodies and DAPI for 2 h at RT, cells were incubated on poly-l -lysine-coated slides and mounted using GB-Mount (GBI Labs) with coverslips. For retina immunofluorescence, paraffin sections of retina were dewaxed with xylene three times, hydrated, and heated in a pressure cooker for epitope retrieval. Tissues were then permeabilized, blocked, and incubated with primary and secondary antibodies. Subsequently, slides were mounted with coverslips using a DAPI-containing mounting medium. Anti-GHRH Ab (1:100, Abcam, ab187512), anti-GHRH-R Ab (1:100, Abcam, ab76263), FITC-conjugated anti-CD4 Ab (1:50, BioLegend, RM4-5), anti-rabbit secondary Ab (1:100, Alexa Fluor 488/594, Invitrogen), DAPI (1 µg/ml, Invitrogen). Images were captured with a Nikon A1MP confocal microscope using the NIS-Elements software (Nikon). The fluorescence intensity was quantified using ImageJ (National Institutes of Health).
5
Subcellular Localization of CLN5 Variants
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A total of 8 × 104 HeLa cells were seeded on 35 mm glass bottom culture dishes and transfected with 1μg plasmids encoding wtCLN5 or mtCLN5 per-well. Twenty four hours later, transfected cells were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton-X100 in PBS both for 15 min at room temperature. After three washes with PBS, cells were blocked by 3% BSA in PBS and then incubated with a mouse anti-HA tag IgG and a rabbit monoclonal antibody against LAMP-1 both at a dilution of 1:100 for 1 h at room temperature. Following three washes, cells were subsequently incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 564-conjugated goat anti-mouse IgG at a dilution of 1:200 for 1 h at room temperature. Cell nuclei were stained with DAPI. After extensive washes, stained cells were analyzed under Nikon A1 MP confocal microscope equipped with a 60 × oil immersion objective. Image sequences were processed and analyzed using NIS-elements Viewer software (Nikon).
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