Confocal laser scanning microscopy was performed with a Leica TCS SP5 SMD confocal system (Leica Microsystems). The images were captured using a single z step, and the emitted fluorescence was detected by scanned detectors at 490–520 and 575–605 nm and emission filters. Predefined settings for the laser power and detector gain were used for all experiments. Microphotographs were analysed using LAS AF version 2.2.1 (Leica Microsystems) or Volocity 6.01 software.
Las af version 2
Manufactured by Leica
Sourced in United Kingdom, Germany
LAS AF version 2.2.1 is a software application for image acquisition and analysis. It provides a user interface for controlling and configuring Leica microscopes and related hardware.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp5 smd confocal system, by Leica (4 mentions)
Tcs sp5 laser scanning system, by Leica (2 mentions)
Metamorph premier version 7, by Molecular Devices (2 mentions)
Ab16502, by Abcam (2 mentions)
Tcs sp5, by Leica (2 mentions)
Anti lamin a c, by Santa Cruz Biotechnology (1 mentions)
Sc 7292, by Santa Cruz Biotechnology (1 mentions)
Sc 166583, by Santa Cruz Biotechnology (1 mentions)
Plan apo, by Leica (1 mentions)
Anti fibrillarin, by Abcam (1 mentions)
Sp5 x, by Leica (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Anti oct4, by Santa Cruz Biotechnology (1 mentions)
Tcs sp2 spectral confocal microscope, by Leica (1 mentions)
Aleuria aurantia lectin, by Vector Laboratories (1 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (1 mentions)
13 protocols using las af version 2
1
Confocal Microscopy Imaging Protocol
2
Live-Cell Confocal Microscopy Imaging
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Confocal Laser Scanning Microscopy (CLSM) was performed with a Leica TCS SP5 SMD confocal system (Leica Microsystems). For live imaging, temperature was maintained at 37°C with 5% CO2 using an environmental control chamber (Life Imaging Services and Solent Scientific). Images were acquired, using a 63X objective, at time points noted in the figures or figure legends and analyzed using the LAS AF version 2.2.1 (Leica Microsystems) or Volocity 6.01 software.
3
Live-Cell Confocal Microscopy Imaging
4
Real-Time Confocal Imaging of Live Cells
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Confocal Laser Scanning Microscopy (CLSM) was performed with a Leica TCS SP5 SMD confocal system (Leica Microsystems). For live imaging, the temperature was maintained at 37 °C with 5% CO2 using an environmental control chamber (Life Imaging Services and Solent Scientific). Images were acquired using a 63X objective at time points noted in the figures or figure legends and analyzed using the LAS AF version 2.2.1 (Leica Microsystems) or Volocity 6.01 software.
5
Immunofluorescence Imaging of IAV Infection
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Cultured A549 cells were infected with the IAV WSN strain (MOI of 5 PFU/cell). At 9 hpi, cells were fixed with 3.7% formalin (20 min, room temperature) and stored in PBS. For immunofluorescence, cells were permeabilised in PBS containing 0.5% Triton X-100 (5 min) and incubated with the following primary antibodies diluted in PBS/0.1% BSA (w/v): rabbit anti-hCLE (1:1000)22 (link), rat polyclonal anti-NP (1:2000)27 (link), monoclonal anti-PA (1:2)66 (link), monoclonal anti-Rab11 (BD Biosciences), and monoclonal anti-influenza HA antibody (1:3) generously provided by J.A. Melero (ISCIII, Madrid, Spain). Confocal microscopy was performed with a Leica TCS SP5 laser scanning system. Images of 1024 × 1024 pixels and an eight bit gray scale depth were acquired sequentially every 0.2–0.3 μm using LAS AF version 2.2.1 software (Leica) and analysed using LAS AF and MetaMorph Premier version 7.5.2 image analysis software (Molecular Devices). For colocalisation analyses, single confocal sections and the colocalisation mask that produces binary images showing only overlapping pixels (white spots) were used.
6
Quantifying p65 Nuclear Translocation
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Cells were fixed in 4% paraformaldehyde (20 min, room temperature) and stored in PBS. For immunofluorescence, cells were permeabilized (5 min) in PBS containing 1% Triton X-100 and incubated with primary antibodies diluted in PBS/4% BSA (w/v) as follows: rat anti-NP (1:500053 (link);, anti-p65 ab16502 (1:500; Abcam) and anti-lamin A/C (636)(1:200; sc-7292, Santa Cruz) to label nuclear envelope. Confocal microscopy was performed with a Leica TCS SP5 laser scanning system. Images of 1024 × 1024 pixels and an eight-bit grayscale depth were acquired sequentially every 0.2–0.3 μm using LAS AF version 2.2.1 software (Leica) and analyzed using LAS AF and MetaMorph Premier version 7.5.2 image analysis software (Molecular Devices). p65 nuclear translocation was quantified by counting at least 200 cells/condition, and the ratio of relative p65 intensity in nucleus and cytoplasm of each cell was calculated.
7
Immunofluorescence Imaging of NP, p65, and MAVS
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Cells were fixed in 4% paraformaldehyde (10 min, room temperature) and stored in PBS. For immunofluorescence, the cells were permeabilized (10 min) in PBS containing 1% Triton X-100 and incubated with primary antibodies diluted in PBS/4% BSA (w/v) as follows: rat anti-NP (1:5000; [24 (link)]), rabbit anti-p65 (ab16502) (1:500; Abcam, Cambridge, United Kingdom and mouse anti-MAVS antibody (E-3): sc-166583 (1:500, Santa Cruz, Dallas TX, USA)). Confocal microscopy was performed with a Leica TCS SP5 laser scanning system (Leica Microsystems, Wetzlar, Germany). Images of 1024 × 1024 pixels with eight-bit grayscale depth were acquired sequentially every 0.2–0.3 μm using LAS AF version 2.2.1 software (Leica) and analyzed using LAS AF and MetaMorph Premier version 7.5.2 image analysis software (Molecular Devices, ForteBio, Wokingham, Berkshire UK).
8
Live-Cell Imaging of Stress Granules
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U2OS cells expressing mCherry-G3BP1 and GFP-LAMP1 were incubated with 2 mM LLOMe for live-cell fluorescence image, which was performed using an inverted microscope (confocal TCS SP5, Leica, LAS AF version 2.6.0), a 63× PlanAPO oil-immersion objective lens (NA 1.4). Two-color time-lapse images were acquired at 340 ms intervals and z-stacks collapsed into 2D projections to generate movies.
9
Live-cell imaging of BAPTA-AM and LLOMe in HeLa cells
10
FRAP Microscopy for Cytoplasmic Protein Dynamics
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The fluorescence recovery after photobleaching (FRAP) experiments were performed using the argon laser (488 nm) of a Leica TSC SP-5 X confocal microscope (Leica Microsystems, Mannheim, Germany). The objective magnification was 64× and the numerical aperture was 1.4. Live cells were kept in a cultivation hood (EMBL, Heidelberg, Germany) to maintain optimal cultivation conditions, (including optimal humidity, 37°C, and 5% CO2) up to the time of visualization. Visualization of GFP- and YFP-fluorescence was performed with the white-light laser.
Leica software (LEICA LAS AF, version 2.1.2) was used to monitor live cells as described by Orlova et al.36 (link) The time-lapse scanning mode was used to monitor cell motion and movement of PRMT1 cytoplasmic bodies. Images of the live cells were captured every 10 s at a resolution of 1024×1024 pixels and a speed of 400 Hz. The bidirectional scanning mode was used for time-lapse microscopy, and LEICA LAS AF software was used for analysis of fluorescence intensity. Statistical analysis (Student’s t-test) was performed with SigmaPlot software, version 8.0.
Leica software (LEICA LAS AF, version 2.1.2) was used to monitor live cells as described by Orlova et al.36 (link) The time-lapse scanning mode was used to monitor cell motion and movement of PRMT1 cytoplasmic bodies. Images of the live cells were captured every 10 s at a resolution of 1024×1024 pixels and a speed of 400 Hz. The bidirectional scanning mode was used for time-lapse microscopy, and LEICA LAS AF software was used for analysis of fluorescence intensity. Statistical analysis (Student’s t-test) was performed with SigmaPlot software, version 8.0.
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