Samples were imaged with a Leica TCS SP5 SMD inverted confocal microscope (Leica Microsystems AG, Vetzlar, Germany) interfaced with Ar, DPSS and HeNe lasers for excitation at 488, 560 and 633 nm, respectively. Live cells were mounted in a thermostated chamber at 37 °C (Leica Microsystems, Vetzlar, Germany) and viewed with a 40 × 1.5 NA oil immersion objective (Leica Microsystems, Vetzlar, Germany) with pinhole aperture set at 1.0 Airy. All images were analyzed using FiJi software (version 2.0.0) and colocalization was determined with JACoP plugin.
Tcs sp5 smd
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 SMD is a confocal laser scanning microscope designed for high-resolution imaging. It features a modular design and integrated scanning system to provide accurate and reliable data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Thermostated chamber at 37 c, by Leica (2 mentions)
1.5 na oil immersion objective, by Leica (2 mentions)
Goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Cy3 dye, by Jackson ImmunoResearch (1 mentions)
Uplsapo, by Olympus (1 mentions)
Fv1000 confocal module, by Olympus (1 mentions)
4 protocols using tcs sp5 smd
1
Live-cell Confocal Imaging Protocol
2
Confocal Microscopy of Cells
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Cells were imaged using a Leica TCS SP5 SMD inverted confocal microscope (Leica Microsystems AG, Wetzlar, Germany) interfaced with a diode laser (Picoquant, Berlin, Germany) for excitation at 405 nm, with Ar lasers for excitation at 488 and 561 nm. Glass-bottom Petri dishes containing cells were mounted in a thermostated chamber at 37 °C (Leica Microsystems) and viewed with a 63 × 1.2 NA water immersion objective or 40× 1.5 NA oil immersion objective (Leica Microsystems). The pinhole aperture was set to 1.0 Airy. All data collected were analyzed by ImageJ software version 1.44o.
3
Localization of TC0668 Protein via Confocal Microscopy
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The localization of the TC0668 protein was analyzed using laser confocal microscopy with a Leica TCS SP5 SMD at 24 h post-infection using specific mouse antibodies (anti-HSP60, anti-PGP3 (primary antibodies)), and a goat anti-mouse IgG (secondary antibody) conjugated to Cy3 dye (red, Jackson Immuno-Research Laboratories) with co-staining with anti-rabbit TC0668 antibody (primary antibody) and goat anti-rabbit IgG (secondary antibody) conjugated to 488 dye (green; Jackson Immuno-Research Laboratories) mixed with DAPI dye (for nuclear staining).
4
Confocal and Two-Photon Imaging of Neurons
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Optical sections (512 × 512 pixels) were acquired with a confocal microscope (Leica TCS SP5 SMD on an inverted DM6000 microscope) using an oil objective HCX PL APO CS 40 × (numerical aperture NA = 1.25), and pinhole was set to 1.47 AU. Digital zoom was adjusted for sampling spines correctly. For whole-cell reconstruction, z-stacks were acquired every 0.5 μm. Sequential illumination with HeNe 633, Ar 561, Ar 488, Ar 458, and diode (Picoquant, Berlin, Germany) 405 laser lines was used for Alexa647, TRITC and Cherry, EGFP and Alexa488, Turquoise2 and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Neurons in Fig. 1a and Supplementary Fig. 1 were acquired with the same acquisition parameters.
For two-photon uncaging, images were acquired using an Olympus FV1000 confocal module on an inverted IX81 microscope with immersion oil objective UPLSAPO 60× (NA = 1.35) and pinhole was set to 180 μm. Digital zoom was set to 8x. Used laser lines were Ar 488 and HeNe 543 for EGFP and Cherry excitation, respectively. For two-photon uncaging, 720 nm line was set on a tunable Chameleon Vision II Ti:Sapphire pulsed laser (Coherent, 80 MHz). Green and red channels were acquired before 720 nm stimulation (−5′ time point) and 60′ after medium change (see the following two-photon uncaging section).
For two-photon uncaging, images were acquired using an Olympus FV1000 confocal module on an inverted IX81 microscope with immersion oil objective UPLSAPO 60× (NA = 1.35) and pinhole was set to 180 μm. Digital zoom was set to 8x. Used laser lines were Ar 488 and HeNe 543 for EGFP and Cherry excitation, respectively. For two-photon uncaging, 720 nm line was set on a tunable Chameleon Vision II Ti:Sapphire pulsed laser (Coherent, 80 MHz). Green and red channels were acquired before 720 nm stimulation (−5′ time point) and 60′ after medium change (see the following two-photon uncaging section).
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