The images were captured using confocal microscopes. Notably, epifluorescent microscopes can also be successfully used to create high quality images suitable for the segmentation pipeline (not shown here, see Lignell et al., 2017 (link)). All embryonic mouse kidney samples were mounted in 99.5% glycerol on glass slides with coverslip spacers (Invitrogen) and imaged using a Leica TCS SP8 X confocal microscope (63x oil-immersion objective, NA 1.4). The chick neural tube images were acquired by using the Andor Dragonfly Spinning disc confocal microscope (63x water immersion NA 1.7). The epithelial spheroids were imaged by using a Nikon A1R + confocal microscope (40x oil-immersion objective, NA 1.3.)
Dragonfly spinning disc confocal microscope
Manufactured by Oxford Instruments
Sourced in United Kingdom
The Dragonfly Spinning disc confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy. It utilizes a spinning disc to provide fast, high-resolution, and low-phototoxicity imaging of live samples.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8 x confocal microscope, by Leica (2 mentions)
Coverslip spacers, by Thermo Fisher Scientific (2 mentions)
A1r confocal microscope, by Nikon (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 donkey anti goat, by Thermo Fisher Scientific (1 mentions)
Cldn1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Apc conjugated cd31 antibody, by BioLegend (1 mentions)
A31572, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
Donkey serum, by Jackson ImmunoResearch (1 mentions)
Hoechst 33342 b2261, by Merck Group (1 mentions)
Ab27591, by Abcam (1 mentions)
Cellsens standard, by Olympus (1 mentions)
Fusion software, by Oxford Instruments (1 mentions)
Ab104139, by Abcam (1 mentions)
Smz1500, by Nikon (1 mentions)
Ni u fluorescent microscope, by Nikon (1 mentions)
Ultraview vox, by Oxford Instruments (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
15 protocols using dragonfly spinning disc confocal microscope
1
Confocal Microscopy for Imaging Embryonic Tissues
2
Multiplex Immunostaining of Kidney Organoids
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Fixed kidney organoids were incubated in blocking buffer (PBS 1X donkey serum 10% triton X100 0.3%) at 4°C for 3h before adding primary antibodies against HNF4α (Life Technologies 1:300, cat# MA1-199), Nephrin (NPHS1 1:300, Bioscientific, cat# AF4269) and Claudin-1 (CLDN1 1:100, Thermo Fisher Scientific, cat# 71-7800) at 4°C for 2 days. After washing in PBS 1X triton X-100 0.1%, organoids were incubated in secondary antibodies 1:400 at 4°C for 2 days: Alexa fluor 405 donkey anti-mouse (Abcam, cat# ab175659), Alexa fluor 488 donkey anti-goat (Molecular Probes, cat# A11055) and Alexa fluor 568 donkey anti-rabbit (Life Technologies, cat# A10042). Samples were then washed before blocking at 4°C for 3h with PBS 1X mouse serum 10μg/ml triton X-100 0.3% and adding an APC-conjugated CD31 antibody (1:50, Biolegend, cat# 303115) at 4°C for 2 days. Finally, samples were washed and imaged in 50:50 glycerol:PBS 1X using a Dragonfly Spinning Disc Confocal Microscope (Andor Technology).
3
Immunofluorescence Staining of Ovarian Tissue
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Immunostaining was performed according to previously described protocols (60 (link)). Briefly, the collected ovaries were fixed with 4% (w/v) PFA for 12 to 24 hours, dehydrated, embedded in paraffin, and cut into 8-μm sections. After deparaffinizing and gradually hydrating the sections, they were put into 0.01% sodium citrate buffer (pH 6.0) and then subjected to microwave antigen retrieval for 16 min. The sections were blocked with 10% donkey serum (Jackson ImmunoResearch) at room temperature for 1 hour, and then the tissue sections were incubated with primary antibodies overnight at 4°C. Next, the sections were thoroughly washed in PBS, incubated with the secondary antibody at room temperature for 1 hour, then stained with Hoechst 33342 (B2261, Sigma-Aldrich) for 1 min. The main primary antibodies used in immunofluorescence are as follows: goat anti-FOXL2 (1:300 dilution; IMG-3228, Novus Biologicals) and DDX4 (1:300 dilution; ab27591, Abcam). Second antibody dilution: donkey anti-goat Alexa Fluor 488 (1:150 dilution; A11055, Invitrogen) and donkey anti-rabbit Alexa Fluor 555 (1:150 dilution; A31572, Invitrogen). Images were acquired on a Nikon Eclipse Ti digital fluorescence microscope, Leica (DMi8), or Andor Dragonfly spinning-disc confocal microscope. The image data were analyzed by the software ImageJ. Image merging was by using the color-merge channels function in ImageJ.
4
Confocal Microscopy for Imaging Embryonic Tissues
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The images were captured using confocal microscopes. Notably, epifluorescent microscopes can also be successfully used to create high quality images suitable for the segmentation pipeline (not shown here, see Lignell et al., 2017 (link)). All embryonic mouse kidney samples were mounted in 99.5% glycerol on glass slides with coverslip spacers (Invitrogen) and imaged using a Leica TCS SP8 X confocal microscope (63x oil-immersion objective, NA 1.4). The chick neural tube images were acquired by using the Andor Dragonfly Spinning disc confocal microscope (63x water immersion NA 1.7). The epithelial spheroids were imaged by using a Nikon A1R + confocal microscope (40x oil-immersion objective, NA 1.3.)
5
Confocal Microscopy Imaging and Analysis
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Immunolabeled slides, including paraffin sections, cryosections, squash slides, and chromosome spreads, were imaged by confocal microscopy (Dragonfly Spinning Disc confocal microscope driven by Fusion Software, Andor Technology, Belfast, UK). Projection images were then processed and analyzed using Bitplane Imaris (version 9.7) software. The histological and TUNEL-stained samples were imaged using an epifluorescence microscope (BX52, Olympus) equipped with a digital camera (DP80, Olympus) and processed using cellSens Standard (Olympus) software packages.
6
Oocyte Maturation in Female Mice
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Female mice (n = 3 for each genotype) at 3 weeks of age were intraperitoneally injected with 5 IU pregnant mare’s serum gonadotropin (PMSG) (Ningbo Sansheng Pharmaceutical) followed by human chorionic hormone (hCG) (Ningbo Sansheng Pharmaceutical) 44 hours later. Oocytes were collected from the oviducts 14 hours after hCG injection, and the PB1 emission was observed with a stereoscope (Nikon, SMZ1500). For IF staining, oocytes were fixed in 4% PFA for 30 minutes at room temperature following permeabilization in PBS containing 0.5% Triton X-100 for 30 minutes. Oocytes were then blocked in PBS containing 1% BSA for 1 hour and incubated with anti–α-tubulin–FITC antibody (1:500 dilution, MilliporeSigma, catalog F2168) for 1.5 hours at room temperature. After washing in PBS, oocytes were shifted onto glass slides, and the slides were sealed with Mounting Medium with DAPI (Abcam, catalog ab104139). Images were acquired with a Dragonfly spinning disc confocal microscope (ANDOR Technology). The experiments were performed independently in triplicate.
7
Microscopic Imaging of Synchronized Neurons
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Animals were synchronized at specific stages. Larva and adult animals were immobilized using 50 mM muscimol on 3% agarose pad. For examining AIY Zone 2 morphology, we used the Nikon Ni-U fluorescent microscope with FITC filter and 40× objectives. All images presented in this study and for fluorescent intensity quantification were taken with Perkin Elmer UltraView VoX or Andor Dragonfly Spinning Disc Confocal Microscope with 40× objectives and 488 nm (for GFP) or 561 nm (for mCherry or RFP) laser. Images were displayed as extended focus projection. We used Adobe photoshop CC to process the rotation and brightness/contrast levels.
8
Optimized Immunostaining of Kidney Organoids
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Fixed kidney organoids were immunostained as previously described,27 (link) with antibodies listed in Table 3 and DAPI (1:1000, Molecular Probes, Waltham, MA; D1306) increasing incubation times to up to 96 hours to ensure antibody penetration in subcellular structures. For labeling of primaries raised in the same species, a four-step immunofluorescence protocol was used following the manufacturer’s instructions (www.jacksonimmuno.com ). Briefly, incubation with first primary antibody was followed by incubation with a specific Alexa Fluor 647–conjugated monovalent Fab fragment (Table 3 ) in excess, thus effectively blocking the first primary antibody. The second primary was then incubated followed by an appropriate secondary antibody. Nonspecific overlapping detection of antigens was controlled for all experiments before inclusion in final analysis.
Samples were imaged in 50:50 glycerol:PBS 1× using a Dragonfly Spinning Disc confocal microscope (Andor Technology, Belfast, UK) fitted with an Andor Zyla camera using 20× (CFI Plan Apochromat Lambda 20×, N.A. 0.75; MRD70200) or 100× (CFI Plan Apochromat Lambda 100×, N.A. 1.45; MRD01905) objectives.
Samples were imaged in 50:50 glycerol:PBS 1× using a Dragonfly Spinning Disc confocal microscope (Andor Technology, Belfast, UK) fitted with an Andor Zyla camera using 20× (CFI Plan Apochromat Lambda 20×, N.A. 0.75; MRD70200) or 100× (CFI Plan Apochromat Lambda 100×, N.A. 1.45; MRD01905) objectives.
9
Confocal Imaging of Fluorescent Proteins
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Confocal images were acquired with an Andor Dragonfly Spinning Disc Confocal Microscope with 40x or 60x objectives. The fluorescently tagged fusion proteins GFP or mCherry was imaged with 488 or 561 nm excitation wavelength lasers, respectively. Animals were anesthetized with 50mM muscimol or Polybead Microspheres 0.10μm (the recorded about GCaMP and PHluorin). Images were processed with Imaris, ImageJ (Fiji) and Photoshop. All images are oriented anterior to the left and dorsal up.
10
Visualization of Cortical Granules in Oocytes
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To detect the cortical granules, oocytes of the NG and the SMG groups at the GV stage were fixed in 4% PFA in PBS for 15 min at room temperature (25 °C), followed by treatment with 0.5% Triton X-100 for 20 min. Oocytes were subsequently incubated in PBS supplemented with 1 mg/mL BSA (Sigma-Aldrich, V900933) for 1 h. After staining with LCA (Lens Culinaris Agglutinin)-FITC for 2 h (1:100 dissolved in PBS, L32475, ThermoFisher Scientific) at room temperature, the oocytes were washed three times in PBS and imaged using an Andor Dragonfly spinning-disc confocal microscope as previously described64 (link). All steps were at room temperature (25 °C).
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