106 T-cells in 400 µl of XFSFM were treated with anti-CD3-targeted NPs containing 3 µg cy5-labeled eGFP mRNA for 1 h at 4 °C for surface binding, followed by a 2-h incubation at 37 °C for internalization. Following these treatments, the cells were washed 3 times with cold PBS, and loaded onto poly-l-lysine (Sigma)-coated slides for 30 min at 4 °C. The samples were fixed in 2% paraformaldehyde, mounted in ProLong Gold Antifade reagent (Invitrogen), and imaged with a Zeiss LSM 780 NLO laser scanning confocal microscope.
Lsm 780 nlo confocal laser scanning microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 780 NLO is a confocal laser scanning microscope developed by Zeiss. It is designed for high-resolution imaging of biological samples, providing superior optical sectioning and contrast enhancement capabilities. The instrument utilizes a combination of laser excitation and advanced detection technologies to enable detailed visualization of cellular structures and dynamic processes.
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Lab products found in correlation
Axio observer z1 inverted microscope, by Zeiss (2 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
D4008, by Merck Group (1 mentions)
Concanamycin a, by Merck Group (1 mentions)
Zen 2010 b sp1, by Zeiss (1 mentions)
Axiovision, by Zeiss (1 mentions)
Accuri c6, by BD (1 mentions)
Plan apochromat 63 1.4 oil dic, by Zeiss (1 mentions)
Plan apochromat 63x 1.4 oil dic m27, by Zeiss (1 mentions)
Vectashield containing dapi, by Vector Laboratories (1 mentions)
9 protocols using lsm 780 nlo confocal laser scanning microscope
1
T-Cell Internalization of Targeted NPs
2
Ethanol-induced GFP-ATG8e fluorescence and MDC staining in Arabidopsis leaves and roots
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GFP fluorescence in detached 4-week-old rosette leaves of GFP-ATG8e [44 (link)] was observed following treatment with 100 mM ethanol for 6 h; water treatment was used as a control (Mock). An LSM 780 NLO laser-scanning confocal microscope (Carl Zeiss, Germany) was used to observe the signals from GFP-ATG8e fusion protein. GFP fluorescence was detected at 488 nm filtered through a primary dichroic filter (UV/488/543).
MDC staining was carried out as described previously [29 (link)]. Briefly, 7-day-old seedlings were treated with or without light submergence (LS) in a 15-mL centrifuge tube containing 10 mL of sterile water for the indicated time and submersed in PBS buffer (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 [pH 7.4]) plus 0.05 mM monodansylcadaverine (MDC, Sigma-Aldrich) for 10 min. Following two rinses with PBS buffer, MDC-stained mature root cells were observed and photographed under an Axio Observer Z1 Inverted microscope (Carl Zeiss) using the 405 nm laser line.
MDC staining was carried out as described previously [29 (link)]. Briefly, 7-day-old seedlings were treated with or without light submergence (LS) in a 15-mL centrifuge tube containing 10 mL of sterile water for the indicated time and submersed in PBS buffer (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 [pH 7.4]) plus 0.05 mM monodansylcadaverine (MDC, Sigma-Aldrich) for 10 min. Following two rinses with PBS buffer, MDC-stained mature root cells were observed and photographed under an Axio Observer Z1 Inverted microscope (Carl Zeiss) using the 405 nm laser line.
3
Monitoring Autophagosome Formation in Arabidopsis
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The stable transgenic lines expressing a GFP-ATG8e fusion and MDC staining were used to monitor autophagosome formation.35 (link) Seven-d-old GFP-ATG8e seedlings grown in culture vessels (7 × 10 cm) were submerged with sterilized water with or without 0.5 µM Concanamycin A (Sigma, C9705). Primary root cells were observed using an LSM 780 NLO laser scanning confocal microscope (Carl Zeiss, Germany) with a 40× oil immersion objective lens. GFP fluorescence were excited by a wavelength of 488 nm and detected at 493 to 558 nm.
For MDC staining, 7-d-old seedlings were not treated or submergence-treated for 24 h and subsequently immersed in phosphate-buffered saline (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 pH 7.4) plus 0.05 mM monodansylcadaverine (MDC; Sigma, D4008) for 10 min. After 2 washes with phosphate-buffered saline, the root cells were observed using an Axio Observer Z1 Inverted microscope (Carl Zeiss, Germany) with a 4',6-diamino-phenylindole–specific filter.
For MDC staining, 7-d-old seedlings were not treated or submergence-treated for 24 h and subsequently immersed in phosphate-buffered saline (135 mM NaCl, 4.7 mM KCl, 10 mM Na2HPO4, 2 mM NaH2PO4 pH 7.4) plus 0.05 mM monodansylcadaverine (MDC; Sigma, D4008) for 10 min. After 2 washes with phosphate-buffered saline, the root cells were observed using an Axio Observer Z1 Inverted microscope (Carl Zeiss, Germany) with a 4',6-diamino-phenylindole–specific filter.
4
Quantifying TAMRA+ Cells in Human Gliomas
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To analyze the percentage and distribution of TAMRA+ cells in human gliomas, 1 million freshly isolated cells were incubated in 1 mL DMEM premixed with 0.5 μg TAMRA-labeled AluDNA for 1 hour at room temperature in the dark. The cells were spun down by brief centrifugation, washed once with the medium, and resuspended in the final volume of 200 μL DMEM. Cell suspensions were layered onto the glass slides using cytospin apparatus (1000 rpm, 1 minutes) and mounted in a droplet of ∼10 μL antifade (DABCO) +0.5 μg/mL DAPI. The slides were covered with coverslips and analyzed under fluorescent microscope AxioVision (Zeiss, Germany) using ISIS V. 5.4.9 (MetaSystems) software for imaging or under LSM 780 NLO confocal laser scanning microscope (Zeiss, Germany) and ZEN 2010 B SP1 software.
5
Cellular Uptake of DOX-Loaded Nanoparticles
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The cellular uptake of different DOX-loaded nanoparticles was examined by confocal laser microscopy and flow cytometry. Before confocal laser microscopy imaging or flow cytometry analysis, RAW-264.7 and MDA-MB-231 cells were grown on glass coverslips in 24-well plates (or cultured in 6-well culture plates for flow cytometry analysis) overnight. Then the cells were incubated with free DOX, DOX/MSNs, RM-(DOX/MSNs) or iRGD-RM-(DOX/MSNs) at an equivalent DOX concentration of 10 μg/mL for 0.5 hours or 1 hour. For confocal laser microscopy imaging, the cells were fixed with immunostaining fixative for 15 min, permeabilized with 0.1% Triton X-100 for 5 min, incubated with phalloidin-F488 for 30 min and then counterstained with DAPI for 5 min at 25 °C. Images were taken with an LSM780NLO confocal laser scanning microscope (Zeiss, Germany). For flow cytometry analysis, the cells were collected after the administration of each treatment, centrifuged at 1500 rpm for 5 min, and washed three times with PBS (pH 7.4). After that, cells were suspended in 300 μL of PBS and analyzed by flow cytometry (Accuri C6, BD, American). For the blocking test, cells were preincubated with free iRGD for 30 min, after which the iRGD was removed, and the cells were incubated with iRGD-RM-(DOX/MSNs).
6
Imaging Microtubule Dynamics in Root Hairs
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Microtubule dynamics were analyzed by high-frequency time-lapse imaging of seedlings of icr2-2×UBQ10::RFP-MBD×ICR2-3×YPet at 8 DAG. Seedlings were grown on CellView 35/10 mm glass-bottomed cell culture dishes (Greiner, 627860) at a 45° angle, so that roots grew along the glass bottom between the growth medium and the glass. Imaging of root hairs was done by taking a z-stack of ten focal planes at 5-min intervals, for a total of 30 frames, using an LSM 780-NLO confocal laser scanning microscope (Zeiss) with a 63× water immersion objective. Images were de-noised using ND-Safir software (Boulanger et al., 2010 (link)).
7
Multiscale Imaging of Cellular Processes
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For RRS, images of the cells were obtained using an Andor spinning disk: Olympus IX 83 inverted microscope, equipped with a Yokaga CSU-X1 Spinning disk Unit and BOREALIS technology for homogeneous illumination. The acquisition software is IQ3.
For RNAFish, UDS, TCR-UDS, and IF of splicing complex after local damage, images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective: Plan-Apochromat ×63/1.4 oil DIC (Differential Interference Contrast) M27 or ×40/1.3 oil DIC. The acquisition software is ZEN.
PLA and IF associated with PLA have been performed on a Zeiss Z1 imager right using a ×40/0.75 dry objective. The acquisition software is Metavue.
Images of the cells for each experiment were obtained with the same microscopy system and constant acquisition parameters. All images were analyzed with ImageJ software. All experiments have been performed at least two times and are biological replicates.
Error bars represent the standard error of the mean of the biological replicates. Excel was used for statistical analysis and plotting of all the numerical data. Statistics were performed using a Student’s test to compare two different conditions (siMock vs. siRNA X or No UV vs. after irradiation) with the following parameters: two-tailed distribution and two-sample unequal variance (heteroscedastic).
For RNAFish, UDS, TCR-UDS, and IF of splicing complex after local damage, images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective: Plan-Apochromat ×63/1.4 oil DIC (Differential Interference Contrast) M27 or ×40/1.3 oil DIC. The acquisition software is ZEN.
PLA and IF associated with PLA have been performed on a Zeiss Z1 imager right using a ×40/0.75 dry objective. The acquisition software is Metavue.
Images of the cells for each experiment were obtained with the same microscopy system and constant acquisition parameters. All images were analyzed with ImageJ software. All experiments have been performed at least two times and are biological replicates.
Error bars represent the standard error of the mean of the biological replicates. Excel was used for statistical analysis and plotting of all the numerical data. Statistics were performed using a Student’s test to compare two different conditions (siMock vs. siRNA X or No UV vs. after irradiation) with the following parameters: two-tailed distribution and two-sample unequal variance (heteroscedastic).
8
Fluorescence in situ Hybridization Protocol
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Cells were grown on 18 mm coverslips, washed with warm PBS and fixed with 4% paraformaldehyde for 15 min at 37° C. After two washes with PBS, cells were permeabilized with PBS + 0.4 % Triton X-100 for 7 min at 4° C. Cells were washed rapidly with PBS before incubation (at least 30 min) with prehybridization buffer : 15% formamide in 2X SSPE pH8.0 (0.3M NaCl, 15.7mM NaH 2 PO 4 .H 2 O and 2.5mM EDTA). 35 ng of probe was diluted in 70 µl of hybridization mix (2X SSPE, 15% formamide, 10% dextran sulphate and 0.5 mg/ml tRNA). Hybridization of the probe was conducted overnight at 37° C in a humidified environment. Subsequently, cells were washed twice for 20 min with pre-hybridization buffer and once for 20 min with 1X SSPE. After several washing with PBS, the coverslips were mounted with Vectashield containing DAPI (Vector). The probe sequence (5' to 3') is: Cy5-AGACGAGAACGCCTGACACGCACGGCAC. Images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective: Plan-Apochromat 63X/1.4 oil DIC M27.
9
Multi-modal Microscopy Imaging Protocol
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For RRS, images of the cells were obtained using an Andor spinning disk : Olympus IX 83 inverted microscope, equipped with a Yokaga CSU-X1 Spinning disk Unit and BOREALIS technology for homogeneous illumination. The acquisition software is IQ3.
For RNAFish, UDS, TCR-UDS and IF of splicing complex after local damage, images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective:
For RNAFish, UDS, TCR-UDS and IF of splicing complex after local damage, images of the cells were obtained using a Zeiss LSM 780 NLO confocal laser scanning microscope and the following objective:
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