DIC and epifluorescence images of dissociated cells in culture were collected on a Zeiss Axiovert microscope with 40X and 63X objectives. Confocal images were obtained on Leica TCS SP5 and Nikon Eclipse TI confocal microscopes.
Tcs sp5
Manufactured by Nikon
Sourced in Germany
The TCS SP5 is a laser scanning confocal microscope system developed by Nikon. It is designed to capture high-resolution, three-dimensional images of samples. The TCS SP5 utilizes a combination of laser illumination and advanced optics to provide detailed, non-invasive imaging of specimens.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse ti, by Nikon (2 mentions)
Axiovert microscope, by Zeiss (2 mentions)
A8806, by Merck Group (1 mentions)
T8787, by Thermo Fisher Scientific (1 mentions)
C9999, by Merck Group (1 mentions)
Softworx software, by GE Healthcare (1 mentions)
Anti flag m2 antibody, by Merck Group (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
780 confocal microscope, by Zeiss (1 mentions)
Normal goat serum (ngs), by MP Biomedicals (1 mentions)
Deoxycholate acid, by Merck Group (1 mentions)
Mowiol mounting medium, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
9 protocols using tcs sp5
1
Dissociated Cell Imaging Protocol
2
Immunofluorescence Imaging of Cells
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Cells grown over Matrigel‐coated coverslips were fixed with 4% paraformaldehyde (SantaCruz Biotechnology #SC‐281692), permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich #T8787), blocked in PBS (ThermoFisher Scientific #A12856‐01) with 1% bovine serum albumin (BSA, Sigma‐Aldrich #A8806) for 30 minutes at 37°C and incubated with the primary antibody overnight at 4°C. Cells were subsequently incubated with the secondary antibody for 30 minutes at 37°C and mounted with ProLong Gold Antifade Mountant with 4′,6‐diamidino‐2‐phenylindole (DAPI, ThermoFisher Scientific #P36930). Primary and secondary antibodies are listed in Table S1 . For Ki‐67 detection, we first performed an antigen retrieval step in which cells were heated for 10 seconds in a microwave with citrate buffer pH 6.0 (Sigma‐Aldrich #C9999) letting cells cool down 20 minutes. Acquisition of fluorescence images was performed in a Leica TCS‐SP5 or a Nikon Eclipse Ti fluorescence microscope. Images were processed using the Adobe Photoshop CS5 or ImageJ software.28 Positive cells were counted using the ImageJ software from at least three random fields per preparation.
3
Multimodal Imaging of Cellular Structures
4
Measuring Pancreatic Calcium Dynamics
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We transferred individual pancreatic slices to a perifusion system containing 6 mM glucose in HEPES-buffered ECS at 34°C. We show representative traces for each experimental condition. Each of these conditions has been tested on at least three separate experimental days. Slices were exposed to a series of square-pulse-like stimulations characterized by exposure to 8 mM glucose for 25 min, followed by a washout with substimulatory 6 mM glucose concentration until all the activity switched off. Imaging was performed on standard confocal microscopes equipped with resonant scanners (Leica Microsystems TCS SP5 and SP8 or Nikon A1R) both upright and inverted with their respective sets of ×20 high numerical aperture (NA) objective lenses. Acquisition frequency was set to at least 20 Hz at 256 × 256 pixels, with pixels size close to 1 µm2 to allow for a precise quantification of [Ca2+]c oscillations. Calbryte 520 was excited by a 488 nm argon laser (Leica SP5 and Nikon A1R) or 490 nm line of a white laser (Leica SP8). The emitted fluorescence was detected by HyD hybrid detector in the range of 500–700 nm using a photon counting mode (Leica) or GaAsP PMT detectors (Nikon).
5
Visualizing Neuronal Circuits via Fluorescence Imaging
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Brain dissections, fixation, and immunostaining were performed as described (Pitman et al., 2011 (link); Wu and Luo, 2006 (link)). To visualize native GFP fluorescence, dissected brains were fixed in 4% (w/v) paraformaldehyde in PBS (1.86 mM NaH2PO4, 8.41 mM Na2HPO4, 175 mM NaCl) and fixed for 20 min at room temperature. Samples were washed for 3 × 20 min in PBS containing 0.3% (v/v) Triton-X-100 (PBT). The neuropil was counterstained with nc82 (DSHB) or monoclonal anti-FLAG M2 antibody (F3165, Sigma) and goat anti-mouse Alexa 647 or Alexa 546. Primary antisera were applied for 1–2 days and secondary antisera for 1–2 days in PBT at 4˚C, followed by embedding in Vectashield. Images were collected on a Leica TCS SP5, SP8, or Nikon A1 confocal microscope and processed in ImageJ.
APL expression of tetanus toxin was scored by widefield imaging of mCherry. mCherry expression in APL was distinguished from 3XP3-driven dsRed from the GH146-FLP transgene by using separate filter cubes for dsRed (49004, Chroma: 545/25 excitation; 565 dichroic; 605/70 emission) and mCherry (LED-mCherry-A-000, Semrock: 578/21 excitation; 596 dichroic; 641/75 emission).
APL expression of tetanus toxin was scored by widefield imaging of mCherry. mCherry expression in APL was distinguished from 3XP3-driven dsRed from the GH146-FLP transgene by using separate filter cubes for dsRed (49004, Chroma: 545/25 excitation; 565 dichroic; 605/70 emission) and mCherry (LED-mCherry-A-000, Semrock: 578/21 excitation; 596 dichroic; 641/75 emission).
6
Quantitative Imaging of Cardiomyocyte Development
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Embryos were imaged with a Zeiss 780 confocal microscope using a × 20 objective with a dipping lens. Z-stacks were taken every 1 μm. Three-dimensional images were reconstructed with ImageJ software. A Leica TCS SP-5 or a Nikon A1R confocal microscope was used for imaging of histological sections. The percentage of tagBFP cells was quantified using ImageJ considering the area of tagBFP and comparing it with the area of myosin heavy chain (MHC) staining.
The percentage of tbx5a-derived cells was quantified applying a median filter of radius 1 in ImageJ. The GFP+ and MHC+ areas were measured. The same set of images was used to quantify the percentage of GFP+ cardiomyocytes in trabeculae and in the regenerated compact layer, applying the same threshold for both regions.
The percentage of tbx5a-derived cells was quantified applying a median filter of radius 1 in ImageJ. The GFP+ and MHC+ areas were measured. The same set of images was used to quantify the percentage of GFP+ cardiomyocytes in trabeculae and in the regenerated compact layer, applying the same threshold for both regions.
7
Confocal Microscopy for Biological Imaging
8
Brain Immunofluorescence Staining Protocol
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For immunofluorescence, brains were dissected and transferred to a tube containing 100 μl ice cold DPBS solution. After centrifugation at 800 xg for 5 min, the supernatant was replaced by 4% Formaldehyde in PBT 0.3% (DBPS + 0.3% Triton X-100 (Sigma)) and incubated at room temperature with rotation for 15 min. Brains were washed with PBT 0.3% three times, rotating for 10 min at room temperature each time and then blocked in Pax-DG (10 g BSA (Sigma), 3g Deoxycholate Acid (Sigma), 3ml Triton X-100 (Sigma), 50ml Normal Goat Serum (MP Biomedicals), 100ml 10X PBS, 850ml H2O) for 2 hours at room temperature with rotation. Primary antibody mixes were created in Pax-DG (dilutions detailed in the Key Resources Table) and brains were incubated in these mixes overnight at 4°C with rotation. The next day, brains were washed with PBT 0.3% three times, rotating for 10 min at room temperature each time and then stained with secondary antibody mixes in Pax-DG (dilutions detailed in the Key Resources Table) for 2 hours at room temperature with rotation. Brains were washed with PBT 0.3% three times, rotating for 10 min at room temperature each time and mounted in Mowiol mounting medium (Sigma). Imaging was performed using the Leica TCS SP5 and Nikon Spinning Disk confocal microscopes.
9
Evaluating Retinal Degeneration through Confocal Microscopy
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Images of immunolabeled cryosections were acquired by using a Leica TCS SP5 (Wetlzar, Germany) or a Nikon 80i confocal microscope. The central dorsal retina was selected for the analysis, since retinal degeneration develops in that specific region called a hotspot [39 (link)]. The same parameters were set up for all the acquisitions. For the final images, ~22 planes at a distance of 0.5 µm were acquired. The fluorescence intensity of all markers was quantified through ImageJ software. Differences in immunofluorescence signals throughout the retinal layers (OS, ONL, OPL, INL, IPL, GCL) were investigated through ImageJ software and by using profile plots (range 0–50) with the corresponding grayscale intensities. For the LD + 7rec group, the outer retina was indicated as SUB/ONL (subretina/outer nuclear layer) since the degenerated tissue loses the physiological retinal architecture with the occurrence of rosettes and neovascularization from the choroid [26 (link)].
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