Fluorescence imaging was carried out as described previously (Singh and Aballay, 2017) with slight modifications. Briefly, the animals were anesthetized using an M9 salt solution containing 50 mM sodium azide and mounted onto 2% agar pads. The animals were then visualized using a Leica M165 FC fluorescence stereomicroscope. For quantification of intestinal lumen bloating, brightfield images were acquired at each time point using the Leica LAS v4.6 software and the diameter of the intestinal lumen was measured using ImageJ software. For quantification of fluorescent immune reporters, fluorescent images were acquired using the Leica LAS v4.6 software in greyscale as presented, and the fluorescence intensity was measured and averaged across three points in the intestine of each animal at the indicated time points using ImageJ software.
Las v 4
Manufactured by Leica
Sourced in Germany, Switzerland
LAS V.4.4 is a software application developed by Leica for imaging and analysis of microscope samples. It provides a versatile platform for a range of microscopy techniques, including fluorescence, brightfield, and phase contrast microscopy. The software offers tools for image acquisition, processing, and analysis, catering to the needs of researchers and laboratory professionals.
Automatically generated - may contain errors
Lab products found in correlation
Dm6000b, by Leica (14 mentions)
M165 fc, by Leica (6 mentions)
Dfc450c, by Leica (5 mentions)
Dfc450, by Leica (5 mentions)
Dm lb2 microscope, by Leica (5 mentions)
Mc120 hd microscope camera, by Leica (5 mentions)
Dm 1000 led microscope, by Leica (5 mentions)
Dm5500b microscope, by Leica (4 mentions)
Dm5000b microscope, by Leica (4 mentions)
Dfc7000t, by Leica (4 mentions)
Dc500 camera, by Leica (4 mentions)
Hybridization buffer, by Qiagen (4 mentions)
Mc190 hd, by Leica (3 mentions)
Dfc550, by Leica (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Dfc480 camera, by Leica (3 mentions)
Dmc2900 camera, by Leica (3 mentions)
Mc170 hd, by Leica (3 mentions)
Dfc450 camera, by Leica (3 mentions)
Dm4000b, by Leica (3 mentions)
160 protocols using las v 4
1
Fluorescence Imaging of Intestinal Lumen
2
Intestinal Lumen Distention and Bacterial Fluorescence Quantification
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Fluorescence imaging was carried out as described previously (10) . the animals were anesthetized using an M9 salt solution containing 50 mM sodium azide and mounted onto 2% agar pads. The animals were then visualized using a Leica M165 FC fluorescence stereomicroscope. For quantification of intestinal lumen distention, brightfield images were acquired at 24 hr for P. aeruginosa and 4 hr for E. faecalis using the Leica LAS v4.6 software, and the diameter of the intestinal lumen was measured using ImageJ software. For quantification of fluorescent bacteria, fluorescent images were acquired using the Leica LAS v4.6 software, and ImageJ software was used to first draw a region of interest around the bacteria in the intestines of animals and then to measure fluorescence intensity in the region.
3
Fluorescence Imaging of Intestinal Lumen Distention
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Fluorescence imaging was carried out as described previously [10 (link)]. Briefly, the animals were anesthetized using an M9 salt solution containing 50 mM sodium azide and mounted onto 2% agar pads. The animals were then visualized using a Leica M165 FC fluorescence stereomicroscope. For quantification of intestinal lumen distention, brightfield images were acquired at 24 h for P. aeruginosa and 4 h for E. faecalis using the Leica LAS v4.6 software, and the diameter of the intestinal lumen was measured using ImageJ software. For quantification of fluorescent bacteria, fluorescent images were acquired using the Leica LAS v4.6 software, and ImageJ software was used to first draw a region of interest around the bacteria in the intestines of animals and then to measure fluorescence intensity in the region.
4
Intestinal Lumen Distention Imaging
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Fluorescence imaging was carried out as described previously (Singh and Aballay, 2017 (link)) with slight modifications. Briefly, the animals were anesthetized using an M9 salt solution containing 50 mM sodium azide and mounted onto 2% agar pads. The animals were then visualized using a Leica M165 FC fluorescence stereomicroscope. For quantification of intestinal lumen distention, brightfield images were acquired at each time point using the Leica LAS v4.6 software, and the diameter of the intestinal lumen was measured using ImageJ software. For quantification of fluorescent immune reporters, fluorescent images were acquired using the Leica LAS v4.6 software in grayscale as presented, and the fluorescence intensity was measured and averaged across three points in the intestine of each animal at the indicated time points using ImageJ software.
5
Confocal Microscopy Imaging Protocol
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Immunofluorescent imaging was performed using a Nikon Eclipse Ti confocal microscope controlled by NisElements 4.30 software (Nikon). A 60× oil immersion objective (NA = 1.4) or a 40× oil immersion objective (NA = 1.3) was employed. Z-stacks with a step size of 0.5 or 1 μm were acquired unless indicated otherwise.
Immunohistochemical stainings were imaged using a Leica DM2500LF microscope equipped with a 10× air objective (NA = 0.32) controlled by Las V4.12 software (Leica) and a Leica DM5000B microscope equipped with a 60× oil objective (NA = 1.4) controlled by Las V4.4 software (Leica). Single focal plane images were acquired.
In all cases, laser settings were adjusted to prevent saturation in any of the channels. Laser settings were kept constant within independent experiments to allow valid comparison of different conditions. For imaging of GVBs and tau pathology markers in SN or in the α-syn model, a hippocampal slide from a tauopathy patient or neurons from the FTDtau1+2 model, respectively, were taken along to set imaging parameters.
Immunohistochemical stainings were imaged using a Leica DM2500LF microscope equipped with a 10× air objective (NA = 0.32) controlled by Las V4.12 software (Leica) and a Leica DM5000B microscope equipped with a 60× oil objective (NA = 1.4) controlled by Las V4.4 software (Leica). Single focal plane images were acquired.
In all cases, laser settings were adjusted to prevent saturation in any of the channels. Laser settings were kept constant within independent experiments to allow valid comparison of different conditions. For imaging of GVBs and tau pathology markers in SN or in the α-syn model, a hippocampal slide from a tauopathy patient or neurons from the FTDtau1+2 model, respectively, were taken along to set imaging parameters.
6
Quantifying Cell Migration via 2-Well Insert
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Cell migration was observed using a 2-well culture insert (No. 80209, Animalab, Germany). The inserts were placed in 6-well plates uncoated and completely coated with nfND. Colloids were dried under sterile conditions, and then inserts were placed in 6-well plates coated by nfND. Cells were cultivated in a 2-well culture insert in 70 µL of DMEM to achieve a monolayer. According to the manufacturer's information, the growth area in the culture insert was 0.22 cm2 per well and the cell-free gap was 500 µm ± 100 µm. After 24 h of incubation and removal of the insert, the cell layer was washed with PBS to remove any non-adherent cells. The 6-well plate was filled with 2 mL of DMEM in each well. The cultivation time was adjusted to nearly complete cell coverage of the free gap, ie 72 h for HS-5 cells and 7 days for C3A and HepG2 cells. After incubation, cells were stained with haematoxylin-eosin (H+E). Images were acquired in the same area at 10× magnification of the free gap using a digital camera (Leica MC190 HD) mounted on an inverted microscope with LAS V4.10 software (Leica, Wetzlar, Germany). The wound-closure rate was assessed on the basis of extracellular spaces (%) of a 1-mm2 area using ImageJ® 1.48v. software (National Institutes of Health, Bethesda, MD, USA).
7
Morphological Analysis of C60-Cultured Cells
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To assess the morphology of cells grown on the C60 surface, C60 dots and blank wells (control wells) of 6-well plates were examined using an inverted light microscope (Leica, TL-LED, Wetzlar, Germany), connected to a digital camera (Leica MC190 HD), using LAS V4.10 software (Leica, Wetzlar, Germany). The cells were stained using hematoxylin-eosin (H+E).
8
Microscopic Assessment of EMT Induction
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An inverted light microscope (Leica, TL-LED, Wetzlar, Germany) was used to assess the cell morphology and the effectiveness of EMT induction after the addition of growth factors. After incubating cells with growth factors for 48 h, cells were stained with May Grünwald-Giemsa stain. For each group of cells, five photos were taken with a digital camera (Leica MC190 HD) and LAS V4.10 software (Leica).
9
Proliferating Tumor Cell Quantification
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At 48 hpi, embryos were fixed with a 4% PFA solution, and whole-mount immunofluorescence assays were performed as previously described [59 (link)] with the specific monoclonal antibody anti-human Ki-67 (Dako, Agilent Technologies, Santa Clara, CA, USA) and Alexa fluor 555 goat anti-mouse (Molecular Probes, Eugene, OR), for the evaluation of proliferating tumor cells. Briefly, embryos were washed in PBS with 0.1% Tween20, permeabilized for 20 min in cold acetone, incubated in NH4Cl solution for 30 min at room temperature, blocked for 2 h at room temperature in blocking solution (5% bovine serum albumin (BSA) in PBS- 0.1% Tween20) and incubated overnight at 4 °C in anti-human Ki-67 antibody 1:200 in blocking solution. The next day, embryos were washed every hour with blocking solution at room temperature until the incubation overnight at 4 °C in Alexa fluor 555 goat anti-mouse 1:500 in blocking solution. On the last day, embryos were washed in 0.1% Tween20/PBS at room temperature. All images were taken with a Leica M205 FA stereomicroscope equipped with a Leica DFC 450 C digital camera using the LAS V4.2 software (Leica Microsystems, Wetzlar, Germany).
10
Neuroanatomical Mapping of Mouse Brain
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At the end of the behavioral studies, mice were re-anesthetized with Ketamine (35 mg/kg) and chloral hydrate (4%), and perfused transcardially with saline and 4% phosphate-buffered paraformaldehyde. Selected sections (40 µm) including the red nucleus and cerebellar nuclei were mounted on gelatinized glass slides and stained using the Nissl technique with 0.1% cresyl violet, to determine the proper location of stimulating electrodes and recording micropipettes. Photomicrographs were taken with the help of a 5x lens of a Leica DMRE microscope equipped with a Leica DFC550 camera, and with the LAS V4.2 software (Leica Microsystems GmbH, Wetzlar, Germany). Photomicrograph reconstructions were made with the Microsoft Office Professional Plus 2010 and CorelDRAW X4 software.
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