One hundred unembryonated eggs were treated with water, 1% v/v DMSO in water or test compounds at a final concentration of 100 μM (unless stated) with 1% v/v DMSO, in the dark at 26°C, either for 56 days or for shorter periods as described. Images were collected on an Olympus BX63 upright microscope using a 60x / 1.42 PlanApo N (Oil) objective and captured and white-balanced using an DP80 camera (Olympus) in monochrome mode through CellSens Dimension v1.16 (Olympus). Images were then processed and analysed using the image analysis platform Fiji [18 (link)].
Cellsens dimension v1
Manufactured by Olympus
Sourced in Japan, Canada
The CellSens Dimension v1.16 is a software suite for image analysis and quantification in life science applications. It provides tools for acquiring, processing, and analyzing microscopic images. The software supports a range of imaging modalities and is compatible with various Olympus microscope systems.
Automatically generated - may contain errors
Lab products found in correlation
Bx63 upright microscope, by Olympus (3 mentions)
Dp80 camera, by Olympus (3 mentions)
Ix73 inverted microscope, by Olympus (3 mentions)
Superfrost plus slide, by Thermo Fisher Scientific (2 mentions)
Microscope bx55, by Olympus (2 mentions)
Dp73 camera, by Olympus (2 mentions)
Ix73 microscope, by Olympus (2 mentions)
Cytospin 4, by Thermo Fisher Scientific (2 mentions)
Fibronectin, by Merck Group (1 mentions)
μ slide vi0.4 ibitreat, by Ibidi (1 mentions)
Ix83 inverted microscope, by Olympus (1 mentions)
Slide scanner microscope, by Leica (1 mentions)
Aperio imagescope, by Leica (1 mentions)
Pe anti mouse cd31, by BioLegend (1 mentions)
Prolong gold, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Ab186352, by Abcam (1 mentions)
F3165, by Merck Group (1 mentions)
Ab12223, by Abcam (1 mentions)
Ab13259, by Abcam (1 mentions)
18 protocols using cellsens dimension v1
1
Embryo Development Assay Protocol
2
Measuring Immune Cell Adhesion under Flow
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1 × 105 control or ICAM-1 KO E0771 cells were seeded on a μ-SlideVI0.4 ibiTreat (ibidi) previously coated with 5 μg/ml fibronectin (Cat. #F1141, Sigma). A day later, the ibidi slide was connected to a microfluidic flow system. OT-1 CTLs were prepared as described above and perfused in binding medium (Hank’s balanced-salt solution containing 2-mg/ml BSA and 10-mM HEPES pH 7.4, supplemented with 1-mM CaCl2 and 1-mM MgCl2) into the chamber and allowed to settle on the different E0771 cell monolayers for 4 min in the absence of shear flow. Adherent CTLs were subsequently subjected to progressively increasing shear stresses (started at 5 dyn/cm2 and increased to 30 dyn/cm2 with 5-s-long intervals as described (37 (link))). Real time imaging was performed utilizing a phase contrast IX83 Inverted Microscope (Olympus) and CTLs that remained firmly adherent to the E0771 monolayers at the end of the detachment assay were counted using cellSens Dimension v1.16 software (Olympus). The fractions of adherent CTLs out of originally settled CTLs were determined in nine fields of view (664 × 664 μm; 324 × 324 nm/pixel).
3
Quantifying CD8+ T Cell Infiltration
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For mouse tissue, images were collected on an Olympus BX63 upright microscope using a 20×/0.75 UApo/340 objective and captured and white-balanced using a DP80 camera (Olympus) in color mode through CellSens Dimension v1.16 (Olympus). For human tissue, images were acquired on a slide-scanner microscope (Leica) using a 20×/0.30 Plan Achromat objective (Zeiss). Snapshots of the slide-scans were taken using Aperio ImageScope (Leica). Images were then processed and analyzed using Fiji ImageJ (http://imagej.net/Fiji/Downloads ). For mice, 8 fields per region (cortex and choroid) were quantified, for a total area of 2.9 mm2. For each human case, four 4 mm2 fields were quantified in each region, for a total of 16 mm2 per region. Several parameters were measured to assess CD8+ T cell accumulation in each region: absolute number of CD8+ T cells, proportion of vessels with CD8+ T cells, proportion of CD8+ T cells that have transmigrated across the vascular lumen. In human cases both parasitized and non-parasitized vessels were examined. If detected, CD8+ monocytes were not included in counts, and were distinguishable from lymphocytes due to their clearly defined kidney-shaped nuclei. Scoring was performed blinded to CM status by two independent observers, with <10% intra-observer variation found.
4
Murine Brain Immunofluorescence Staining
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Brains were sectioned at 10 µm on a cryostat (Leica), mounted on SuperFrost Plus slides (Thermo Fisher) and fixed with 4% paraformaldehyde for 10 min. Slides were washed with PBS, and non‐specific binding blocked (1% BSA, 0·05% Tween‐20 in PBS) for 1 h, followed by overnight incubation at 4 °C with Alexa Fluor® 647 anti‐mouse CD45·1 (1:100, BioLegend) and PE anti‐mouse CD31 (1:100, BioLegend). Slides were then washed with PBS, incubated with DAPI (0·5 µg/ml) for 30 min, washed again with PBS and covered‐slipped with ProLong Gold (Invitrogen). Images were captured using an Olympus BX63 upright microscope through CellSens Dimension v1·16 (Olympus).
5
Embryonic Development Microscopy Protocol
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One hundred unembryonated eggs were placed in 1ml deionised water in eppendorf tubes in the dark at room temperature, 26°C, 30°C or 37°C either for 56 days or for shorter periods as described. Images were collected on an Olympus BX63 upright microscope using a 60x/1.42 PlanApo N (Oil) objective and captured and white-balanced using a DP80 camera (Olympus) in monochrome mode through CellSens Dimension v1.16 (Olympus). Images were then processed and analysed using the image analysis platform Fiji (18) (link).
6
Immunocytochemistry of SH-SY5Y and HEK293T Cells
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Immunocytochemistry (ICC) was performed as described in Perland et al. (2016) (link) on transfected SH-SY5Y cells and wild-type HEK293T cells (only anti-UNC93A) with antibodies all diluted in anti-UNC93A [rabbit, 1:100ab69443, Abcam, (Ceder et al., 2017 (link))], anti-KCNK3 [mouse, 1:100, ab186352 Abcam, (Schmidt et al., 2015 (link))], anti-KCNK9 (mouse, 1:100, Sigma, K0514), and anti-Flag [mouse, 1:200, F3165, Sigma, (Sriramachandran et al., 2019 )]. Images were acquired using Olympus microscope BX55 with an Olympus DP73 camera and the cellSens Dimension v1.14 (Olympus) and were then handled using ImageJ, Fiji edition (Schindelin et al., 2012 (link)). The epitope against UNC93A was pair-wise aligned against the three isoforms of CG4928 (FBpp0074118, FBpp0074119, and FBpp0309738) using EMBOSS Needle (global alignment) and EMBOSS Water (local alignment) to investigate protein identity and similarity (Li et al., 2015 (link)) (Supplementary Dataset S4 ).
7
Immunocytochemical analysis of UNC93A in mouse cortical neurons
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Wildtype male and female mice were mated and at e14-15 the females were euthanized with cervical dislocation, the embryos removed and cortex dissected out and primary cultures were set up as previously described in Perland et al. (2016) (link). Immunocytochemistry was performed as described in Perland et al. (2016) (link) with anti-UNC93A diluted 1:100 in supermix blocking solution. Co-staining with neuronal marker Pan diluted 1:200 (MAB2300, Millipore), KDEL markers (ab12223, Abcam) diluted 1:200, Syntaxin 6 (Ab12370, Abcam) diluted 1:100, Synaptotagmin (ab13259, Abcam) diluted 1:200 and SNAP25 (ab25737, Abcam) diluted in 1:100 in supermix blocking solution. Images were acquired at the SciLifeLab BioVis Facility (Uppsala University) using confocal LSM710 SIM (Zeiss) and the Zen black software (Zeiss) or Olympus microscope BX55 with an Olympus DP73 camera and the cellSens Dimension v1.14 (Olympus). Images were then handled using ImageJ, Fiji edition (Schindelin et al., 2012 (link)).
In addition, immunocytochemistry for the N-terminal anti-UNC93A antibody (ab173552, Abcam), diluted 1:80 in 5% milk block (Bio-Rad), was performed as described above.
In addition, immunocytochemistry for the N-terminal anti-UNC93A antibody (ab173552, Abcam), diluted 1:80 in 5% milk block (Bio-Rad), was performed as described above.
8
GFP Expression in Malpighian Tubules
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Adult flies were dissected to study the gene expression pattern in Malpighian tubules. To induce CG4928 cells to express green fluorescent protein (GFP), the Pin/Pin-UAS-GFP and the enhancer trap CG4928-GAL4 lines were crossed. Flies were fixed in 4% formaldehyde for 1 h, followed by washing five times in 1 × phosphate-buffered saline with Tween-20 (PBST; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, and 0.1% Tween-20, Sigma Aldrich). Dissection was performed in 1 × PBST on silicon-coated petri dishes. Tissues were mounted on Superfrost Plus slides in 50% glycerol solution. Images were acquired with an Olympus microscope BX55 with an Olympus DP73 camera and the cellSens Dimension v1.14 (Olympus). Images were then handled using ImageJ, Fiji edition (Schindelin et al., 2012 (link)).
9
Fluorescence Imaging of Probes in Cells
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All fluorescence microscopy images and corresponding brightfield images were obtained on an Olympus IX73 microscope. All probe 2 cell images were obtained using a filter cube consisting of 380–420 nm excitation bandpass filter, a 484 nm dichroic mirror, and a 513–604 nm emission bandpass filter. Cell images of 3 were acquired using a filter cube comprising 503–557 nm excitation bandpass filter, a 580 nm dichroic mirror, and a 600–700 nm emission bandpass filter. All cells were plated in 8-chambered wells (Lab-Tek® Chambered #1.0 Borosilicate Coverglass System) at a density of 5 × 104 cells per well one day before imaging. The cells were washed once with DPBS, and were incubated in Opti-MEM with or without 100 μM LPA for 1 h. 20 μM probe 2 was then diluted into the appropriate wells and the cells were incubated for 3 h. The cells were washed five times in DPBS and were imaged in DPBS. All images were analysed using Olympus CellSens Dimension V1.18. H&E images were acquired from a Nikon Eclipse 50i Microscope with a Nikon DS-Ri1 camera and NIS-Elements BR3.0 software.
10
Vimentin Immunofluorescence Imaging Protocol
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Cells were fixed with methanol for 5 min at −20°C, washed three times with PBS, and permeabilized with Saponin in PBS for 5 min. Cells were then blocked in PBS supplemented with 5% BSA. Vimentin rabbit monoclonal D21H3 antibody (dilution 1:200; 5741; Cell Signaling Technology) and Alexa Fluor 568 goat anti-rabbit IgG (H + L; dilution 1:1,000; A11011; Invitrogen) were applied to cells and incubated at RT for 2 h. All images were acquired by Olympus software CellSens Dimension v1.18 on an Olympus SpinSR10 Ixplore spinning disk confocal microscope equipped with an UplanApo 100×/1.5 oil objective (Olympus). The pixel size was optimized to achieve the maximum resolution, which was calculated to be 65 nm.
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