Live worms, colorimetric WISH stains, and whole-worm H3P stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH stains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. H3P cell counts were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/ ) with the cell counter function, and muscle lesions were labelled by hand, using control RNAi animals as a reference so as to only label muscle gaps larger than those found in controls, and quantified using the ImageJ area measurement function. Piwi-1+ and H3P+ cell counts were performed in cross-section using Imaris (Bitplane, South Windsor, CT, USA). Cross-sections were taken from the same regions in control and knockdown animals (examples shown in Fig 3A ), and “normal” and “ectopic” regions were determined manually using DAPI staining to mark the boundary (as shown in Fig 3B ). Collagen+ cells, TUNEL+ cells, and neoblast subclass markers were also quantified using Imaris. Significance was determined by a two-tailed unequal variance student’s t-test. All images were post-processed in a similar manner using Adobe Photoshop. Heatmaps were made in R studio using data downloaded from https://compgen.bio.ub.edu/PlanNET/planexp [61 (link)].
Dmire2 inverted fluorescence microscope
Manufactured by Hamamatsu Photonics
The DMIRE2 is an inverted fluorescence microscope manufactured by Hamamatsu Photonics. It is designed to observe fluorescently labeled samples. The DMIRE2 features a motorized focusing system and a high-performance optics system for high-quality imaging.
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Lab products found in correlation
Back thinned em ccd camera, by Hamamatsu Photonics (5 mentions)
Spinning disc head, by Yokogawa (2 mentions)
Back thinned electron multiplying charge coupled device camera, by Yokogawa (2 mentions)
M165 fluorescent dissecting microscope, by Leica (1 mentions)
Gentamicin, by Wisent (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Annexin 5 alexa fluor 647 conjugate, by Thermo Fisher Scientific (1 mentions)
Volocity 6, by PerkinElmer (1 mentions)
8 protocols using dmire2 inverted fluorescence microscope
1
Quantifying Planarian Regeneration Markers
2
Visualizing Listeria Invasion in HeLa Cells
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HeLa cells were plated at 2×105 cells per well in 6-well tissue culture plates with glass coverslips 48 h prior to infection. At 24 h prior to infection, cells were transfected with LifeAct-RFP. Cells were then infected with wild type Lm expressing GFP (DP-L1039) at an MOI of 100 in DMEM. After 60 min of invasion at 37°C, cells were washed three times with phosphate buffered saline (PBS) with Calcium and Magnesium (Wisent #311-420-CL) followed by the addition of growth media containing 50 μg/ml Gentamicin (Wisent #400-130-IG). At 6 h post infection, coverslips were washed with PBS with Calcium and Magnesium (Wisent #311-420-CL) and transferred into spaceships and RPMI-1640 (Wisent #350-025-CL) with 10% heat-inactivated FBS (Wisent), 50 μg/ml Gentamicin, 2.5 mM CaCl2 and 2% (v/v) Annexin V Alexa Fluor 647 Conjugate (Invitrogen). HeLa cells were maintained at 37°C during imaging. A Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera and spinning disk confocal scan head with a 63X objective and LSM 510 software was used. Volocity software (Improvision) was used to acquire images.
3
Spinning Disk Confocal Microscopy Protocol
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Unless otherwise indicated, cells were imaged using a Quorum spinning disk microscope with a ×63 oil immersion objective (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera or Hamamatsu CMOS FL-400 camera, spinning disk confocal scan head) and Volocity 6.3 acquisition software (Improvision)). Confocal z-stacks of 0.3 μm were acquired and images were analyzed with Volocity 6.3 software.
4
Multimodal Imaging and Quantification of Worm Cells
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WISH, dFISH, and immunostaining were performed as previously described (Currie et al., 2016 (link)). Colorimetric WISH stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH and fluorescent mouse-anti-PIWI-1 (gift from Dr. Jochen Rink) stains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. Cell counts and co-localizations were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/ ). Significance was determined by a two-tailed Student’s t-test. All experiments were, at minimum, triplicated and at least five worms were used per stain and per time point. All labeling images were post-processed using Adobe Photoshop.
5
Time-lapse imaging of Listeria infection
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BMDMs were plated as a monolayer at 4×106 cells/well onto coverslips in a 6-well tissue culture plate. After 24 h, the monolayer was infected with Lm expressing RFP (DP-L5538) at an MOI of 0.01 for 1 h. Cells were washed three times with PBS and incubated at 37°C in RPMI containing 10% FBS, 10% L-929 conditioned medium and 50 μg/mL gentamycin. At 6 h post infection, the coverslips were imaged in 25 mM Hepes buffered RPMI containing 10% FBS and 50 μg/mL gentamycin using a Quorum spinning disk confocal microscope (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu back-thinned electron multiplying charge-coupled device camera, Yokogawa spinning disc head, and Volocity 6 software). Coverslips with BMDM from control and TIM4−/− mice were placed side by side on a dual chamber heated stage at 37°C. Over the course of 12 h, 36 μm z-stacks with a 2 μm step were taken every 15 min at ten foci of infection per coverslip. The channels for DIC and red fluorescence were acquired throughout the experiment. Image analysis was performed to measure the number of infected cells per infection foci during the course of the experiment.
6
Time-lapse imaging of Listeria infection
7
Confocal Microscopy Imaging Workflow
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Unless otherwise indicated, cells were imaged using a Quorum spinning disk microscope with a 63x, 1.4 NA oil immersion objective (Leica DMIRE2 inverted fluorescence microscope equipped with either a Hamamatsu Back-Thinned EM-CCD camera or Hamamatsu CMOS FL-400 camera) and Volocity 6.3 software (PerkinElmer). Confocal z-stacks of 0.3 μm were acquired. Images were analyzed with the Volocity software and then imported into and assembled in Adobe Illustrator for labelling.
8
Competitive Rab7 Binding and Nucleotide Exchange Assay
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When indicated, cells were imaged using a Quorum spinning disk microscope with a 633 oil immersion objective (Leica DMIRE2 inverted fluorescence microscope equipped with a Hamamatsu Back-Thinned EM-CCD camera, spinning disk confocal scan head, and Volocity acquisition software;
(F) Competitive in vitro binding assay. Rab7-bound resin, preloaded with GTPgS, was incubated with equivalent amounts of GST-RILPC33 and increasing concentrations of SopD2. Elutions were analyzed by western blotting. The ratio of RILPC33:SopD2 (1:X) is indicated (one asterisk). (G) Nucleotide exchange assay. Rab7 was loaded with Mant-GDP. Following desalting, exchange reactions were performed supplemented with SopD2 or BSA at the indicated ratios. Reactions were monitored by spectrofluorometry (excitation at 355 nm, emission at 448 nm). Improvision). Confocal z-stack images with z steps of 0.3 mm were acquired, and confocal images were analyzed with Volocity software.
(F) Competitive in vitro binding assay. Rab7-bound resin, preloaded with GTPgS, was incubated with equivalent amounts of GST-RILPC33 and increasing concentrations of SopD2. Elutions were analyzed by western blotting. The ratio of RILPC33:SopD2 (1:X) is indicated (one asterisk). (G) Nucleotide exchange assay. Rab7 was loaded with Mant-GDP. Following desalting, exchange reactions were performed supplemented with SopD2 or BSA at the indicated ratios. Reactions were monitored by spectrofluorometry (excitation at 355 nm, emission at 448 nm). Improvision). Confocal z-stack images with z steps of 0.3 mm were acquired, and confocal images were analyzed with Volocity software.
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