To monitor the cell-cycle, cell division and cell movement, GFP-labeled primary mesothelial cells from CR−/− and WT mice were infected with a lentivirus coding for mCherry-hCdt1 (30/120) [18 (link), 19 (link)]. The Fucci (Fluorescent, ubiquitination-based cell cycle indicator) mCherry-hCdt1 was a kind gift of Prof. H. Miyoshi (Riken, Japan). mCherry-hCdt1 was synthetized by PCR with the forward primer (FW-PmeI-mCherry 5’-AGT CGT TTA AAC ATG GTG AGC AAG GGC GAG GAG-3’) and reverse primer (RV-SpeI-mCherry 5’-AGT CAC TAG TTT AGA TGG TGT CCT GGT CCT G-3’) and cloned into pLVTHM (Addgene plasmid #12247) (SpeI and PmeI sites) substituting eGFP. pLV-mCherry-hCdt1 was used to produce lentivirus. The expression level of mCherry-hCdt1 is cell cycle-dependent showing an accumulation during the G0/G1 phase, followed by an ubiquitination–based protein degradation during S/G2/M phases. Fluorescence and time-lapse microscopy was performed with an inverted fluorescence microscope DMI 6000B (Leica Microsystems) equipped with an incubation chamber. A digital camera (Leica), a 10× objective, GFP and TXR filter sets and the LAS-AF imaging software (Leica) were used to acquire the images. Images were taken every hour using the same settings including exposure times, gains and positions. For the image analysis a Java ImageJ plugin: CGE [17 (link)] was used.
Las af imaging software
Manufactured by Leica
Sourced in Germany
LAS AF is an imaging software developed by Leica for advanced fluorescence microscopy. It provides comprehensive tools for acquiring, processing, and analyzing microscopic images. The software supports a wide range of Leica microscopes and cameras, enabling users to capture high-quality images and perform essential image analysis tasks.
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Lab products found in correlation
Sp5 confocal microscope, by Leica (3 mentions)
Tcs sp5 confocal microscope, by Leica (2 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Syto9 live dead baclight bacterial viability kit, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 conjugated goat anti human kappa f ab 2 antibody, by Southern Biotech (2 mentions)
Hcx pl apo cs 63 1.40 0 60 oil objective, by Leica (2 mentions)
Digital camera, by Leica (1 mentions)
Dmi6000b, by Leica (1 mentions)
Pdl 800 d, by PicoQuant (1 mentions)
Tcs sp5, by Leica (1 mentions)
Maitai, by Spectra-Physics (1 mentions)
Tcs sp5 system, by Leica (1 mentions)
Normal goat serum (ngs), by Jackson ImmunoResearch (1 mentions)
Alexa488 anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
El6000, by Leica (1 mentions)
Tcs spe dmi4000, by Leica (1 mentions)
Sp5 2 photon microscope, by Leica (1 mentions)
Dfc295, by Leica (1 mentions)
Application suite version 3, by Leica (1 mentions)
17 protocols using las af imaging software
1
Cell Cycle Dynamics Visualization
2
Imaging and Analysis of Single- and Two-Cell Clones
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Single cell clones (SCC’s) and two cell clones (TCC’s) were imaged in multiple focal planes, and were displayed as two-color image stacks (one color for the Flp-Out GFP, and one color for anti-discs large or neuroglian-GFP) in Leica’s LAS AF imaging software for the SP5 confocal microscope. Analysis was performed by hand; cells having ambiguous polygonal topology were not counted. To control for the possibility of cell sorting, SCC’s and/or TCC’s were not considered for analysis unless they were separated by at least two cell diameters within the tissue. In order to control for boundary effects, cells located on tissue folds close to the anterior-posterior (AP) or dorsal-ventral (DV) compartment boundaries were not counted. To prevent mis-identification of SCC or TCC clones, we did not consider cells for scoring if the source of the GFP signal was ambiguous (for example, if the GFP source overlapped with another bright clone in a different focal plane).For display (non-analytical) purposes, Figure 1 D shows mitotic cells that have been first inverted, and then subjected to a brightness threshold cutoff in Adobe Photoshop.
3
Two-color STED Microscopy of COS7 Cells
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A STED microscope (TCS SP5; Leica) equipped with a HCX Plan Aprochromat 100×, 1.4 NA oil STED objective and operated with the LAS AF imaging software (version 2.7.3.9723; Leica) was used for performing two-color STED microscopy. Chromeo494 and Atto647N were excited with pulsed diode lasers (PDL 800-D; PicoQuant) at 531 and 640 nm, respectively. The STED beam was generated by a Ti:Sapphire laser (Mai Tai; Spectra-Physics) tuned at 750 nm. The same microscope was used for acquiring confocal images of COS7 cells using an HCX Plan Apochromat 63×, 1.4 NA oil immersion objective.
4
Multi-Marker Imaging of Cell Dynamics
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Confocal microscopy used a Leica TCS SP5 system and imaging processing analysis used Leica LAS AF imaging software. For Ring1b and Tunel detection, argon laser 488 nm line excitation and emission maximum at 525 nm were used. For EdU, BrdU, and annexin V excitation, the argon laser 561 nm was used for excitation and, at 617 nm, used as maximum emission. DAPI/Hoechst 33342 was detected using violet (405 nm) laser line and emission maximum at 470 nm. LacZ sections images were obtained on a Zeiss microscope.
5
Immunolabeling and Confocal Imaging of Adult Brains
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Adult brains were dissected in phosphate buffered saline (PBS), fixed for 15 min in 4% paraformaldehyde, washed in PBT (0.5% Triton X-100 in PBS), stained in primary antibodies at 4°C for ca. 48 h, washed in PBT, stained in fluorescent secondary antibodies at 4°C for overnight, then washed in PBT and mounted using Vectashield (Vector Laboratories, Inc.). A Leica TCS SP5 confocal microscope was used to capture sequential images of the brains at 20× magnification with LAS AF imaging software from Leica (version 2.6.0). Anti-TH mouse monoclonal antibody used in these experiments was obtained from Immunostar (Hudson, WI, USA), and was used at a concentration of 1:500. Anti-GFP rabbit polyclonal antibody was procured from Santa Cruz Biotechnology, Inc., and was also used at a concentration of 1:500. Alexa633 anti-mouse antibody and alexa488 anti-rabbit antibody (Invitrogen) were both used as secondary antibodies at 1:200. All antibodies were diluted in PBT+10% normal goat serum (NGS Jackson ImmunoResearch Laboratories).
6
High-Resolution Imaging of Neuronal Structures
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The immunostained rat hippocampal neurons (Fig 4 , Appendix Figs S12–S14 ), mouse inner hair cells (Appendix Fig S17 ), and neuromuscular junctions (Appendix Fig S18 ), as well as the transferrin and LysoTracker uptake (Appendix Figs S2, S3 and S11 ) and the immunostained GFP‐tagged proteins (Appendix Fig S10 ) were imaged using a pulsed STED microscope, built on the basis of the TCS SP5 confocal microscope (Leica). The microscope was equipped with a pulsed diode laser (18 mW, 80 MHz, 640 nm emission, PicoQuant) for excitation of the STED dye, and with a pulsed infrared titanium: sapphire (Ti:Sa) tunable laser (1W, 80 MHz, 720–1,000 nm, Mai Tai Broadband; Spectra‐Physics) for depletion set at a wavelength of 750 nm. For confocal imaging, an Argon laser (488 nm) and HeNe laser lines (543, 594, 633 nm) were used for excitation. Detection was achieved by ultra‐sensitive avalanche photodiodes and high sensitivity, low noise PMTs (Leica). All samples were imaged using a 100× HCX PL APO oil immersion STED objective (NA 1.4). Images were acquired using the Leica LAS AF imaging software, with a pixel size of 20 × 20 nm, 30 × 30 nm or 60 × 60 nm and a scanning speed of 1,000 Hz.
7
Quantifying Autophagy and Mitophagy via Microscopy
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Images were captured using an inverted microscope (Leica Microsystems TCS SPE DMI4000, Wetzlar, Germany) attached to an external light source for enhanced fluorescence imaging (Leica EL6000) with Leica LAS AF imaging software. Each dataset was imaged in a single session, with the same imaging settings maintained throughout the session. Image analyses were performed in a user-blinded manner using ImageJ and de-noised using a background subtraction rolling ball radius of 50 pixels. For display, images were adjusted for brightness and contrast, with channel minimum and maximum values kept consistent between images. Maximum projections were used for analysis.
To quantify autophagy, high-resolution z-stacks imaged at 40× resolution with an optimal step size were performed to capture multiple cells across the coverslip. Regions of interest (ROIs) were drawn around single cells in ImageJ and selected in the 568 nm channel containing the LC3 signal. The fluorescence intensity of LC3-RFP was measured above a set threshold to exclude background pixel values. To quantify mitophagy, both LC3-RFP and Mitotracker were measured above a set threshold to exclude background pixel values, and the percentage of LC3 puncta that colocalized with mitochondria was calculated for each cell.
To quantify autophagy, high-resolution z-stacks imaged at 40× resolution with an optimal step size were performed to capture multiple cells across the coverslip. Regions of interest (ROIs) were drawn around single cells in ImageJ and selected in the 568 nm channel containing the LC3 signal. The fluorescence intensity of LC3-RFP was measured above a set threshold to exclude background pixel values. To quantify mitophagy, both LC3-RFP and Mitotracker were measured above a set threshold to exclude background pixel values, and the percentage of LC3 puncta that colocalized with mitochondria was calculated for each cell.
8
Quantification of GABApre Synaptic Proteins in SMA
9
Live Imaging of C. elegans Embryo Division
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For AB–P1 division measurements, embryos were obtained by dissecting worms microinjected with MOs and then mounted in egg buffer (Edgar, 1995 (link)). Live imaging was performed at room temperature at 18–28 h after microinjection, with a Leica SP5 inverted scanning laser microscope and 63×1.2 NA Olympus water immersion objective, using DIC settings. Images were acquired with Leica LAS AF imaging software as z-stacks of nine levels taken every ∼12 s for durations that covered the time from P0 cytokinesis until the four-cell stage. Images were processed using ImageJ. Timing of nuclear envelope breakdown (NEBD) (measured at disappearance of nuclear membranes) and cytokinesis (measured at onset of cortical furrowing) in P0, AB and P1 blastomeres was determined as described previously (Benkemoun et al., 2014 (link); Brauchle et al., 2003 (link); Edgar and McGhee, 1988 (link)).
10
Quantification of GABApre Synaptic Proteins in SMA
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Images of synaptic terminals were acquired on a Leica SP8 confocal microscope using a 40× objective with 5× digital zoom at a 2048 × 2048 optical resolution. Acquisition settings for excitation and fluorescence detection parameters were identical for each genotype, SMA controls and SMA+AAV9-BDNF. The GAD65 and GAD67 terminals were analyzed within a 10 μm confocal z-stack at 300 nm step intervals. Surface area and staining intensities were determined using Leica LAS AF imaging software. Relative synaptic protein levels were quantified by assessing the mean gray values, defined as the sum of the gray values of all the pixels in a region of interest (pixel sum), divided by the number of pixels in that region (pixel count), as we reported recently32 (link). Regions of interest were defined as the outline of positively stained terminals. To quantify the relative levels of GABApre synaptic proteins (GAD65 and GAD67), only varicosities directly juxtaposed to proprioceptive afferent terminals (VGluT1+) were considered.
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