The mitochondrial membrane potential (MMP) was measured using JC-1 (5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide), which exhibits potential-dependent accumulation in mitochondria according to the manufacture instruction (MitoProbe™ JC-1 Assay Kit (M34152), ThermoFisher Scientific, U.S.). The JC-1 dye loading solution was diluted into a final concentration of 2 μM. Then, the cells were co-incubated with JC-1 dye at 37°C with 5% CO2 for 30 min. Then the medium was removed and cells were washed three times with PBS. The cells were supplemented with an antifade mounting medium with DAPI. Images were acquired using the OLYMPUS FV1000 inverted confocal microscope (Japan).
Fv1000 inverted confocal microscope
Manufactured by Olympus
Sourced in Japan
The FV1000 inverted confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a sensitive detector, high-resolution optics, and a flexible configuration to support a variety of sample types and research needs.
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Lab products found in correlation
Fluoromount g, by Southern Biotech (2 mentions)
Aperio versa 200, by Leica (2 mentions)
Poly d lysine substrate, by Merck Group (2 mentions)
Prolong diamond antifade, by Thermo Fisher Scientific (2 mentions)
Mitoprobe jc 1 assay kit, by Thermo Fisher Scientific (1 mentions)
Cellrox oxidative stress reagent, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Sa00013 1, by Proteintech (1 mentions)
Sa00013 4, by Proteintech (1 mentions)
Alexa fluor 488 conjugated anti mouse igg antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Abn1660, by Merck Group (1 mentions)
Smi 32, by BioLegend (1 mentions)
Ab144p, by Merck Group (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Thapsigargin, by Thermo Fisher Scientific (1 mentions)
Ab150157, by Abcam (1 mentions)
Fluoview viewer software, by Olympus (1 mentions)
41 protocols using fv1000 inverted confocal microscope
1
Mitochondrial Membrane Potential Assay
2
Measuring Cellular Oxidative Stress with CellROX and MitoSOX
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The CellROX™ Deep Red reagent is a novel fluorogenic probe for measuring cellular oxidative stress which is to be fluorescent upon oxidation by reactive oxygen species. The cytoplasmic ROS was measured using CellROX® Deep Red Reagent according to the manufacturer’s instructions (CellROX® Oxidative Stress Reagents (C10422), ThermoFisher Scientific, U.S.). To detect the mitochondrial superoxide, we used the MitoSOX which could selectively detect the superoxide in the mitochondria of live cells (Kalyanaraman et al., 2012 (link); Forman et al., 2015 (link)) (MitoSOX™ Red mitochondrial superoxide indicator (M36008), ThermoFisher Scientific, U.S.). Briefly, cells were incubated with the CellROX®/MitoSOX Reagent at a final concentration of 5/10 μM for 30 min at 37°C, then removed medium and washed cells three times with PBS. The cells were supplemented with an antifade mounting medium with DAPI. Images were acquired using the OLYMPUS FV1000 inverted confocal microscope (Japan).
3
Confocal Immunofluorescence Analysis of Mitophagy
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For confocal immunofluorescence analysis, cells were fixed in 4% formaldehyde for 10 min, then permeated in 0.2% Triton X-100 for 5 min and blocked in 3% BSA for 2 h. The incubation condition was as follows: CoraLite®488-conjugated TOM20 Monoclonal antibody (CL488-66777, Proteintech, China, 1:200 dilution), CoraLite®594-conjugated LAMP1 Monoclonal antibody (CL594-67300, Proteintech, China, 1:200 dilution), MTCO2 Mouse Monoclonal Antibody (A-6404, ThermoFisher Scientific, U.S., 1:200 dilution) and Parkin Polyclonal Antibody (PA5-13399, ThermoFisher Scientific, U.S., 1:50 dilution) at 4°C over-night. After being washed three times with TBST, the cells were co-incubated with/without fluor-conjugated goat anti-rabbit secondary antibody (red) (SA00013-4, Proteintech, China, 1:200 dilution) and fluor-conjugated goat anti-mouse secondary antibody (green) (SA00013-1, Proteintech, China, 1:200 dilution). The cells were supplemented with an antifade mounting medium with DAPI. Images were acquired using the OLYMPUS FV1000 inverted confocal microscope (Japan). Mitophagy was measured by the co-expression fluorescence of yellow dots (LAMP1 and Tom20 overlay) per field.
4
Immunolabeling and Confocal Analysis of Human Retinal Ganglion Cells
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After electrophysiologic recordings, hRGCs were fixed with 4% paraformaldehyde overnight at 4°C and immunolabeled with the following primary antibodies: RNA-binding protein with multiple splicing (RBPMS, GTX118619,1:200, Genetex, Irvine, CA) postsynaptic density 95 (PSD-95, APZ-009, 1:400, Alomone Labs, Jerusalem, Israel), and AnkG (338800, 1:250, Invitrogen, Carlsbad, CA). Human RGCs were then incubated with appropriate secondary antibodies for 2 hours at room temperature (1:200; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and cover slipped with Fluoromount G (Southern Biotech, Birmingham, AL). We imaged hRGCs with an Olympus FV-1000 inverted confocal microscope. Images were analyzed using Fiji ImageJ Version 1.53c. We manually counted the number of primary and secondary neurites. We measured PSD-95 and AnkG immunolabeling in somas and neurites using freehand selection and segmented line tools in ImageJ.
5
Fluorescence Imaging of Pupal Cell Dynamics
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Pupal cultures washed and imaged in HL3 (70 mm NaCl, 5 mm KCl, 20 mm MgCl2, 10 mm NaHCO3, 5 mm trehalose, 115 mm sucrose, 5 mm HEPES, pH 7.2) with calcium (1.5 mm ). Images were taken as a time series on an XY plane at an interval of 4 s using a 40× objective with an NA of 1.4 on an Olympus FV1000 inverted confocal microscope (Olympus Corp.). The raw images were extracted using Fiji (Schindelin et al., 2012 (link)), and regions of interest (ROIs) representing cells were selected using the Time Series Analyzer plugin. Percentage ΔF/F release was calculated as (F0 – Ft)/F0 × 100, where Ft is the fluorescence at time t and F0 is baseline fluorescence corresponding to the average fluorescence over the first 10 frames. The area under the curve from these response curves was calculated from 300 to 900 s using Microsoft Excel (Microsoft) and implementing the trapezoidal rule (Nedelman and Gibiansky, 1996 (link); Qin et al., 2009 (link)) where
Here, t1 = 300 s and t2 = 900 s, and this accounts for both positive and negative AUC measurements. For experiments involving UAS-Shits, a heated microscope stage was used.
Here, t1 = 300 s and t2 = 900 s, and this accounts for both positive and negative AUC measurements. For experiments involving UAS-Shits, a heated microscope stage was used.
6
Phospho-cSrc Staining in Gastroids
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Gastroids were cultured on MatTek dishes (MATTEK Corporation) and stained for phospho-cSrc. In brief, gastroids were washed twice in 1X PBS, and formalin-fixed. Cells were permeabilized using 1X PBS containing 0.1% Triton X-100 (30 minutes, room temperature), and washed three times in 1X PBS. Gastroids were stained with mouse anti-phospho-cSrc (Y 418: Cell Signaling) followed by Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:200, Molecular Probes). Nuclei were stained with DAPI (4 μg/mL). Images were captured using an Olympus FV-1000 Inverted Confocal Microscope. Image acquisition was performed using Fluoview FV10-ASW 1.7 software.
7
FRET Analysis of MeCP2-Importin α5 Interaction
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FRET acceptor photobleaching experiments were performed to study the interaction of MeCP2 with importin α5. Briefly, HEK293 cells were grown in 35 mm cell culture plates and transfected with 1) MeCP2-GFP + importin α5-mScarlet-I in separate plasmids or in the control conditions 2) cytoplasmic-GFP + importin α5-mScarlet-I or 3) PTE (phosphotriesterase)-GFP + importin α5-mScarlet-I; 12 h prior to the experiment cells were split on 24 mm cover glass coated with poly-L-lysine. Imaging was done in PBS solution containing calcium and magnesium (Sigma). Experiments were performed on the Olympus FV1000 inverted confocal microscope described above. GFP was excited with 488 nm laser line and mScarlet-I with 561 nm laser line. The acceptor (mScarlet-I) was bleached with 561 nm laser at maximal power setting. The FRET efficiency (EFRET, %) was analyzed using the Fiji software and calculated as EFRET = (Dpost + Dpre)/Dpost x 100 with D being the donor (GFP) fluorescence before (pre) and after (post) acceptor bleaching.
8
Whole Corneal Fluorescent Imaging
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Fluorescent whole corneal images were taken using a Nikon Ni fluorescent microscope and a 20× objective; whole corneal images were taken en montage. Central corneal stacked images were acquired using an Olympus FV-1000 inverted confocal microscope using a 40× oil objective.
9
Immunocytochemistry of Fixed Cells
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Cells grown on coverslips were fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized using 0.3% Triton X-100, then blocked with 3% normal donkey serum containing 0.1% Triton X-100 in DPBS, before being stained with primary antibodies at 4°C overnight, followed by the appropriate secondary antibodies for 1 hr at room temperature and 1 μg/mL DAPI (Sigma) for 10 min. Images were acquired using an Olympus FV1000 inverted confocal microscope.
10
Quantifying Neuronal Subtypes in Htr3a-EGFP Mice
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Images were captured via confocal microscopy using an Olympus FV-1000 inverted confocal microscope. Images were then exported from the FluoView viewing software as.tiff files and assembled in Adobe Photoshop (2014 2.2 release, Adobe Systems Inc.). Due to heterogeneity in expression intensity of the Htr3a-EGFP transgene, images were minimally adjusted for optimal brightness and contrast. Numbers of neurons (Hu+ cells), Htr3a-EGFP+ cells, Fast Blue+ cells, and cells labeled with markers of sensory neurons were manually quantified by visual inspection of assembled confocal stacks. Proportions of neuronal subtypes were calculated as the number of immuno-positive cells for each marker over the total number of Hu+ neurons for each section. Cells were counted from three to six sections of three DRG from each axial level group from four Htr3a-EGFP animals.
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