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14 protocols using las x life science software

1

Immunohistochemical Analysis of ACE2 and TMPRSS2 in NAFLD

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Immunohistochemistry was performed on biopsies obtained from 6 NAFLD patients for which formalin-fixed liver sections were available, using the rabbit anti-human ACE2 (ab108252) and rabbit anti-human TMPRSS2 (ab109131, Abcam, Cambridge, UK), both diluted at 1:6400 according to the manufacturer’s instructions, and developed using DAB (3,3′-Diaminobenzidine, Vector Labs) as chromogen as previously described [22 (link)]. Histological images were taken using the BX41 microscope (Olympus), coupled with Leica LAS X Life Science software.
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2

Multiphoton Imaging of AMP-B Distribution

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The corneal tissue with thin rims of sclera tissue was used to study AMP-B distribution by multiphoton microscopy. Three groups were used to perform this study: (1) treated sclerocorneal tissue with a topical application of 200 µL of AMP-B suspension (1000 µg/mL), (2) sclerocorneal tissue treated with MN-loaded AMP-B, and (3) sclerocorneal tissue free from AMP-B administration. Sclerocorneal tissue of each group was prepared previously, and each one was placed on the microscope glass slide on the Leica upright microscope stand DM6000 (Leica Microsystem, Milton Keynes, UK). The images were taken for each treatment, including control, using Leica-TCS-SP8 MP multiphoton excited fluorescence microscope (Leica Microsystem, Milton Keynes, UK). Images were captured using internal spectral hybrid detectors with a gain of around 100. The emission was detected around 472 nm. The images were analysed using Leica LAS-X life science software. 3D images were reconstructed using the same software.
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3

Intraocular AMP-B Distribution via Multiphoton Microscopy

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In vitro studies 2.2.6.1 Intraocular AMP-B delivery and distribution studies using multiphoton microscopy
The corneal tissue with thin rims of sclera tissue was used to study AMP-B distribution by multiphoton microscopy. Three groups were used to perform this study, (1) treated sclerocorneal tissue with a topical application of 200 µL of AMP-B suspension (1000 µg/mL), (2) sclerocorneal tissue treated with MNs loaded AMP-B, and (3) sclerocorneal tissue free from AMP-B administration. Sclerocorneal tissue of each group was prepared previously, and each one was placed on the microscope glass slide on the Leica upright microscope stand DM6000 (Leica Microsystem, Milton Keynes, UK). The images were taken for each treatment, including control, using Leica-TCS-SP8 MP multiphoton excited uorescence microscope (Leica Microsystem, Milton Keynes, UK). Images were captured using internal spectral hybrid detectors with a gain of around 100. The emission was detected around 472 nm. The images were analysed using Leica LAS-X life science software. 3D-Images were reconstructed using the same software.
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4

Lysosomal Rupture Assay with Acridine Orange

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BMDMs were loaded with 1 μg/mL Acridine Orange for 20 min (Antunes et al., 2001 (link)). Images were acquired using a Leica SP8 confocal microscope (Leica Microsystems) and data processing was performed with the LAS X Life Science software (Leica Microsystems). Additional samples were acquired in a FACSCanto II equipment (BD Biosciences) and lysosomal rupture was followed by the loss of red fluorescence from the acidic lysosomal compartment in the PerCP-Cy5.5 channel. Data were analyzed by FlowJo Software (Tree Star).
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5

GFP-PCO Imaging in Nicotiana benthamiana

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For GFP‐PCO imaging, the abaxial side of leaves from transiently transfected Nicotiana benthamiana (4 weeks old) were analysed with a Leica DM6000B/SP8 confocal microscope (Leica Microsystems) using 488‐nm laser light (20% laser transmissivity), photomultiplier tube detection, and emission light was collected between 491 and 551 nm. Images were analysed and exported using the LAS X life science software (www.leica-microsystems.com), with an unchanged lookup table settings for each channel. Imaging of the ratiometric mClover3‐Ubi‐RAP2.32–70‐mRuby reporter was performed using a Zeiss airyscan 800. Fiji was used to quantify mClover3 and mRuby mean fluorescence intensity in protoplast nuclei.
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6

Confocal Imaging of Redox-Sensitive GFP

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Confocal microscopy analysis was carried out on 2‐wk‐old transgenic chl-roGFP2–, chl-roGFP2-Prx–, chl-roGFP2-PrxΔCR–, and chl-PrxΔCR-roGFP2–expressing plants. Images were captured with a Leica TCS SP8 confocal system (Leica Microsystems) and the LAS X Life Science Software, while using a HC PL APO ×40/1.10 objective. To capture chl-roGFP2 fluorescence, samples were excited at 488 nm and emission was measured at 500 to 520 nm. For chlorophyll fluorescence, excitation was at 488 nm and emission was at 670 nm. Merged images were generated using Fiji software.
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7

Visualizing ASC Specks Formation

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THP-1-ASC-GFP cells and ASC-Citrine BMDMs were acquired in a FACSCanto II equipment (BD Biosciences, Franklin Lakes, NJ, US). Gates for priming and ASC-specks formation were established according to the relative distribution of the cell population in the FITC-W and FITC-A channels (Hoss et al., 2018 (link)). Data were analyzed by FlowJo Software version 10.8.1 (Tree Star, Ashland, OR, US). In addition, ASC-Citrine BMDMs were imaged with a SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany) equipped with a 63 × /1.40 oil objective. Data processing was performed with the LAS X Life Science software (Leica Microsystems).
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8

Immunofluorescence Imaging of Bunyavirus Proteins

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Cells were grown on sterile glass coverslips, fixed with 4% PFA, and washed three times with PBS. Fixed cells were permeabilized and blocked by incubation for 40 min with PBS containing 0.25% saponin and 2% FBS. Cells were incubated for 1 h with primary antibodies diluted 1/200 in PBS with saponin and FBS. The following antibodies were used: a rabbit polyclonal antibody specific for the BUNV nucleocapsid (N) protein [7 (link)], a mouse monoclonal antibody specific for BUNV Gc glycoprotein, kindly provided by Prof. Richard M. Elliott [39 (link)] and a rabbit anti-Giantin polyclonal antibody that is a marker of the Golgi complex (BioLegend, San Diego, CA, United States; 924302). Alexa Fluor–conjugated antibodies (Invitrogen) were used as secondary antibodies diluted 1/500 in PBS with 0.25% saponin and 2% FBS. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) diluted 1/200 in PBS with 0.25% saponin and 2% FBS for 20 min. All incubations were conducted at RT. Coverslips were mounted using Prolong-Gold (Life Technologies, Carlsbad, CA, USA). Confocal microscopy images were acquired using a Leica STELLARIS 5 confocal multispectral microscope equipped with an HCX PL APO 63.0 X/1.4 NA oil objective and LAS X Life Science software (Leica Microsystems).
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9

Dual-channel Confocal Imaging of Chlorophyll

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Images were acquired with a Leica TCS SP8 confocal system (Leica Microsystems) and the LAS X Life Science Software, while using a HC PL APO ×40/1.10 objective. All images were acquired at a 4096×4096-pixel resolution, with emission at 500-520 nm following excitation at 488nm for chl-roGFP2
fluorescence and emission at 670nm following excitation at 488nm for chlorophyll fluorescence. Merged images were generated using Fiji (Image 1.A) software.
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10

Neurite Tracing and Quantification

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Immunofluorescence images were acquired with 20 × or 40 × magnification with a fluorescence microscope (DM6000B, Leica) and LAS X Life science software (Leica) using the same microscope settings (exposure time, gain, lamp intensity, magnification) for each experiment or test series. Images were assessed using the FilamentTracer algorithm of the commercially available IMARIS® software, which allows semi-automatic detection, tracing and measurement of neuronal cells and their processes. The software evaluates neurite features such as neurite area, neurite length, neurite diameter and neurite branches. We calculated neurite markers relative to the number of cell nuclei as ratio of a given neurite marker per nucleus.
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