Preliminary experiments were performed to evaluate the amount of mitochondria in control and treated cells to exclude that possible changes in mitochondrial function could be due to uneven mitochondrial numbers. Briefly, H9c2 cells seeded on glass coverslips were loaded with MitoTracker Deep Red 633 mitochondrial fluorescent dye (0.5 μM, Life Technologies, Carlsbad, CA, USA) dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v), added to the culture medium for 20 min at 37 °C. Cells were fixed in 2% buffered paraformaldehyde for 10 min at room temperature and red fluorescence was analyzed using a Leica TCS SP5 confocal scanning microscope (Leica, Mannheim, Germany) equipped with an argon laser source (excitation λ 633 nm) and a x63 oil immersion objective. Mitochondrial amount was also measured by flow cytometry. Single-cell suspensions were incubated with MitoTracker Deep Red 633 (200 nM) for 20 min at 37 °C and immediately analysed with a FACSCanto flow cytometer (Becton–Dickinson, San Jose, CA). Data were analyzed using FACSDiva software (Becton–Dickinson). Since both methods detected no substantial differences among the different experimental groups, the results of the subsequent experiments to assess mitochondrial function were deemed reliable.
Tcs sp5 confocal scanning microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 is a confocal scanning microscope that enables high-resolution imaging of samples. It features multiple laser lines, a spectral detection system, and advanced scanning capabilities to capture detailed images of specimens.
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Lab products found in correlation
Facscanto flow cytometer, by BD (4 mentions)
H2dcfda, by Thermo Fisher Scientific (3 mentions)
Mitosox, by Thermo Fisher Scientific (3 mentions)
Facsdiva software, by BD (2 mentions)
H2dcfda, by Leica (2 mentions)
Mitosox, by Leica (2 mentions)
H2dcfda, by BD (2 mentions)
Mitosox, by BD (2 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (2 mentions)
Mitotracker deep red 633, by BD (1 mentions)
Alexa 488 conjugated anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Avertin, by Fujifilm (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rom 380, by Yamato Scientific (1 mentions)
Anti p16, by Cell Signaling Technology (1 mentions)
Anti gata4, by Proteintech (1 mentions)
Oil immersion objective, by Leica (1 mentions)
Dmem f12 without phenol red, by Thermo Fisher Scientific (1 mentions)
Jem 1011, by JEOL (1 mentions)
Fluoromax 4 spectrofluorometer, by Horiba (1 mentions)
36 protocols using tcs sp5 confocal scanning microscope
1
Mitochondrial Quantification in H9c2 Cells
2
IGF-1R Localization in HT1080 Cells
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HT1080 cells were plated at a density of 5 × 104 cells per well in a 24-well culture plate containing sterilized glass coverslips and grown for 24 h. Next, cells were treated with 1:500 POE dilution for 0.5, 3, 7 and 16 h, fixed with 2% paraformaldehyde for 5 min and permeabilized with ice cold 50% acetone/50% ethanol solution for 4 min at RT. After PBS washing, cells were blocked in saturated solution (0.5% BSA and 2% gelatin) for 30 min at 37 °C. After 1 h of incubation at 37 °C with a mouse anti-IGF-1R monoclonal antibody (Cell Signaling) diluted to 1:100 in saturated solution, cells were washed with PBS for 30 min under stirring conditions and then incubated with Alexa 488-conjugated anti-mouse secondary antibody (Invitrogen Molecular Probes) diluted to 1:200 in PBS for 1 h at 37 °C in the dark. Finally, cells were washed twice with PBS, and coverslips were placed onto microscope slides using a Fluoromount™ Aqueous Mounting Medium. Fluorescent signals were visualized using a Leica TCS SP5 confocal 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.
3
Quantifying Thalamocortical Axon Innervation
4
Monocyte GATA4 and p16 Expression in SLE
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MACS-isolated monocytes from SLE patients and HC individuals were stimulated with 2’3’-cGAMP for 3 h. Monocytes freshly isolated or stimulated with 2’3’-cGAMP were spun onto a microscope slide using Thermo Shandon Cytospin 4 (Thermo Fisher Scientific). Monocytes were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 in PBS. Nonspecific background staining was prevented by incubation with 5% bovine serum albumin in PBS. Cells were incubated overnight at 4°C with the following primary antibodies: anti-GATA4 (1:100, Proteintech, Rosemont, IL, USA, Cat. #19530) and anti-p16 (1:100, Cell Signaling Technology, Danvers, MA, USA, Cat. #18769), and then washed and incubated for 1 h at room temperature with a secondary antibody, Alexa 647 anti-rabbit IgG (1:1000, Cell Signaling Technology, Cat. #4414). All samples were visualized using a Leica TCS SP5 confocal scanning microscope (Leica, Mannheim, Germany), and images were processed using Leica LASAF software and ImageJ (http://imagej.nih.gov/ij/ ).
5
Quantifying Dendritic Spine Morphology
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MADM brain sections were imaged on a Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Mannheim, Germany). Stacks of 5–15 images taken at 0.6–2 μm intervals were acquired with a 63X oil immersion objective (Leica) with 1.7–2.4X zoom. For each cell, dendritic spines were counted on a segment of apical dendrite at least 40 μm in length. Dendrite width was measured by creating a mask of each dendrite, manually erasing dendritic protrusions from the image, then calculating the average width of the dendrite by dividing the total area of the dendrite by its length.
6
Live-cell Confocal Microscopy Imaging
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Leica SP5 II SMD confocal microscope (Leica Microsystems, Mannheim, Germany) or Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Mannheim, Germany) with a 63 × 1.4 numerical aperture and a oil-corrected objective lens was used for the observation of cells. Fluorescence of mCitrine or mGFP was acquired at 495–570 nm with an excitation wavelength of 488 nm (argon ion laser), and that of mCherry at 610–700 nm with an excitation wavelength of 594 nm (laser diode). During observation, the cells were kept at 37 °C in an air–steam cube incubator in Dulbecco’s Modified Eagle Medium (DMEM-F12; without phenol red) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2% FBS.
7
Comprehensive Characterization of Carbon Dots
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Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images were recorded on JEOL JEM-1011 and JEOL JSM-2100, respectively. The CDs were drop-cast onto 400-mesh carbon-coated Cu grids followed by drying in open air at room temperature. The fluorescence spectra of the CDs were recorded on a FluoroMax-4 spectrofluorometer (Horiba Scientific, Japan). X-ray diffraction (XRD) pattern was performed by using a D8 Advance X-ray diffractometer (Bruker) with Cu Kα radiation. Raman spectra were acquired on a Lab RAM Aramis spectrometer (Horiba Scientific, Japan). Fourier transform infrared (FTIR) spectra were measured on Vector-22 spectrometer (Bruker) ranging from 500 to 4000 cm−1, and the samples were dispersed in KBr pellets. Ultraviolet-visible (UV-vis) absorption of the CDs solution was obtained using an UV-1800(PC) UV-vis spectrophotometer (Mapada). X-ray photoelectron spectroscopy (XPS) measurements were performed on a PHI 5000 VersaProbe spectrometer (UlVAC-PHI, Japan). The fluorescence confocal images were obtained using a Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany). The specimens were excited at 488 nm, and the emission was detected from 500 to 530 nm.
8
Visualizing Leaf Pigment Distribution
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Laser confocal microscopy was performed on a Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Heidelberg GmbH, Mannheim, Germany) to investigate distribution of chlorophyll, carotenoids and anthocyanins in the leaves. Leaf samples were dissected and placed in embedding medium (Tissue-tek OCT compound, Sakura Finetek, CA, USA). A Leica CM1950 cryostat was employed to cut the embedded tissue samples and 10 μm thickness transverse sections were made. The sections were transferred to poly-L-lysine-coated slides for observation. For collecting chlorophyll fluorescence, a filter with 633 nm excitation and 650–700 nm emission was used, and for carotenoid autofluorescence, a filter with 488 nm excitation and 500–600 nm emission bands was used [34 (link)]. Anthocyanins were excited at 543 nm with a helium-neon laser [35 (link)]. DAPI (4′,6′-diamidino-2-phenylindole) was used to counterstain the tissues.
9
Oligomers Effect on Calcium Signaling
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Oligomers of HypF-N (4 μm monomer) were incubated for 1 h at 37°C with shaking in cell culture medium with or without 1 μm or 16 μm of αBc, Clu, or M-TTR and then added to N2a cells seeded on glass coverslips for 60 min at 37°C. Cells were then loaded for 30 min with 4.4 μm fluo3-AM (Life Technologies, Carlsbad, CA, USA) with 0.01% (w/v) pluronic acid F-127 (Sigma-Aldrich, St. Louis, MO, USA) in culture medium at 37°C. The cells were then washed with PBS and fixed for 10 min at room temperature with 2% (v/v) paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS. Finally, the paraformaldehyde solution was removed and the coverslips were washed with PBS and water to be mounted on slides for the confocal microscopy analysis. Cell fluorescence was analyzed by a Leica TCS SP5 confocal scanning microscope (Leica Microsystems, Mannheim, Germany), as previously described (Campioni et al., 2010; Zampagni et al., 2011) .
10
Assessing Mitochondrial Oxidative Stress in Vitiligo
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Keratinocytes from perilesional vitiligo skin were seeded on glass cover slips and loaded with the mitochondrial superoxide-specific fluorescent probe MitoSOX (3 μM) and H2DCFDA (2.5 μM; Invitrogen, Carlsbad, CA, USA) – dissolved in 0.1% DMSO and Pluronic acid F-127 (0.01% w/v) – which was added to cell culture media for 15 min. at 37°C. Cells were fixed in 2.0% buffered paraformaldehyde for 10 min. at room temperature and the H2DCFDA and MitoSOX fluorescence analysed with a Leica TCS SP5 confocal scanning microscope (Mannheim, Germany) equipped with an argon laser for fluorescence analysis. A series of optical sections (1024 × 1024 pixels) 1.0 μm in thickness was taken through the cell depth at intervals of 0.5 μm with a Leica 20× objective and then projected as a single composite image by superimposition. Mitochondrial superoxide and ROS generation were also monitored by flow cytometry: single-cell suspensions were incubated with MitoSOX (0.5 μM) and H2DCFDA (1 μM; Invitrogen) for 15 min. at 37°C and immediately analysed with a FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA).
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