The phenotypes and genotypes of conditional mutant flies were assessed. For yellow , the abdomen of the conditional mutant flies was photographed with a Leica Mz10f. For notch, the wings of the conditional mutant flies were scored and photographed with a Leica Mz10f. For bam and nos , each of the conditional mutant female flies was crossed with three w1118 male flies to test the fertility. The ovaries were scored based on their severity of phenotype and photographed with a Leica Mz10f. For ms(3)k81 and cid , each conditional mutant male fly was crossed with three w1118 female virgin flies to test the fertility. The testes were scored according to their fertility and photographed with a Zeiss Imager.Z2. The testes, tips of testes, seminal vesicles, and mature sperm from all conditional mutant flies and control flies were examined under a microscope with light and 4′,6-diamidino-2-phenylindole (i.e., DAPI) staining. The target genomic region from the conditional mutant flies was amplified by PCR using appropriate primers following a previous protocol (Carvalho et al. 2009 (link)) (Table S4 ). For yellow , the whole fly was used for PCR; for notch, the wing, eye, and body were used for PCR; for bam and nos , the ovary was used for PCR; for ms(3)k81 and cid , the testis was used for PCR. The corresponding PCR products were then sequenced.
Mz10f
Manufactured by Leica
Sourced in Germany, United States, Switzerland, Canada
The MZ10F is a stereo microscope designed for routine use in a laboratory setting. It features coaxial coarse and fine focusing controls, a trinocular viewing head, and a transmitted/incident illumination system. The MZ10F is suitable for a variety of applications requiring low magnification observation and analysis.
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Lab products found in correlation
Dfc310 fx, by Leica (10 mentions)
Dfc420c, by Leica (6 mentions)
Dmc2900, by Leica (5 mentions)
Ms 222, by Merck Group (4 mentions)
Tricaine, by Merck Group (4 mentions)
Pli 90 pico injector, by Harvard Apparatus (3 mentions)
Dfc450c, by Leica (3 mentions)
El6000 light source, by Leica (3 mentions)
Hcimage software, by Hamamatsu Photonics (3 mentions)
Dfc425c, by Leica (3 mentions)
Tamoxifen, by Merck Group (3 mentions)
Las v 4, by Leica (3 mentions)
Low melting point agarose, by Promega (3 mentions)
Alizarin red s, by Merck Group (3 mentions)
Alcian blue, by Merck Group (3 mentions)
Microscalpel, by World Precision Instruments (2 mentions)
Ultraview vox spinning disk confocal microscope, by PerkinElmer (2 mentions)
Zen 2.5 imaging software, by Zeiss (2 mentions)
Nis elements basic research software, by Nikon (2 mentions)
Lsm 800 airyscan, by Zeiss (2 mentions)
176 protocols using mz10f
1
Phenotypic and Genotypic Analysis of Conditional Fly Mutants
2
In Situ Hybridization Protocol for Embryonic Expression
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Whole-mount in situ hybridization was carried out as described previously (Hammes et al., 2001 (link)). In situ hybridization on sections was performed as described previously (Jensen and Wallace, 1997 (link)), except that the signal was enhanced by performing the colour reaction in the presence of 10% polyvinyl alcohol (Sigma Aldrich, P8136). Probe synthesis was conducted with the components of the DIG RNA labelling kit (Roche, 11277073910).
Images of embryos were taken using a Leica MZ 10F stereomicroscope (Leica LAS V4.9 imaging software). Images were processed in ImageJ to isolate specimen and adjust the background colour.
Primers used for cloning templates for in situ riboprobes: Lrp4 ISH probe primer forward: TAC CAT CGA AGC ATC TCG GC, reverse: TTC GTG TTT CCA GCC TGT GT; Lrp5 ISH probe primer forward: ATG CCG GCG GAG TGA AG, reverse: GAG TAG AAA GGC TCC CTC GG.
Lef1 riboprobe kindly provided by Thomas Willnow and Walter Birchmeier, MDC, Berlin, Germany.
Images of embryos were taken using a Leica MZ 10F stereomicroscope (Leica LAS V4.9 imaging software). Images were processed in ImageJ to isolate specimen and adjust the background colour.
Primers used for cloning templates for in situ riboprobes: Lrp4 ISH probe primer forward: TAC CAT CGA AGC ATC TCG GC, reverse: TTC GTG TTT CCA GCC TGT GT; Lrp5 ISH probe primer forward: ATG CCG GCG GAG TGA AG, reverse: GAG TAG AAA GGC TCC CTC GG.
Lef1 riboprobe kindly provided by Thomas Willnow and Walter Birchmeier, MDC, Berlin, Germany.
3
Zebrafish Pronephric Tracer Assay
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Zebrafish were maintained in standard conditions (Westerfield, 2000 ) at the University of Manchester Biological Services Unit according to the UK Animals Act 1986. The ocrl−/− mutant line (ZDB-GENO-120531–1) has been described previously (Ramirez et al., 2012 (link)). WT fish were of AB background.
Lysine-fixable 10-kD dextran labeled with Alexa Fluor 488 (Molecular Probes) was prepared in PBS at 2 µg/µl final concentration. Zebrafish embryos at 72 h after fertilization were treated for 60 min with DMSO control (0.005% DMSO), 5 µM m-3m3fbs, or 5 µM o-3m3fbs by addition to the water. Embryos were then anesthetized with 0.2 mg/ml MS222 (Sigma-Aldrich) in chorion water, and tracer was injected into the common cardinal vein using a glass micropipette PLI-90 Pico-Injector (Harvard Apparatus). Embryos were returned to the respective drug treatments and incubated at 29°C. Pronephric accumulation was assessed 2 h after injection on whole mount embryos using a fluorescent dissecting stereomicroscope (MZ10F; Leica).
Lysine-fixable 10-kD dextran labeled with Alexa Fluor 488 (Molecular Probes) was prepared in PBS at 2 µg/µl final concentration. Zebrafish embryos at 72 h after fertilization were treated for 60 min with DMSO control (0.005% DMSO), 5 µM m-3m3fbs, or 5 µM o-3m3fbs by addition to the water. Embryos were then anesthetized with 0.2 mg/ml MS222 (Sigma-Aldrich) in chorion water, and tracer was injected into the common cardinal vein using a glass micropipette PLI-90 Pico-Injector (Harvard Apparatus). Embryos were returned to the respective drug treatments and incubated at 29°C. Pronephric accumulation was assessed 2 h after injection on whole mount embryos using a fluorescent dissecting stereomicroscope (MZ10F; Leica).
4
GUS Staining of MRG1 and MRG2
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For GUS staining, MRG1 and MRG2 genomic sequences were amplified by primers MRG1-3/MRG1-4 and MRG2-5/MRG2-6, respectively. They were fused to the GUS genomic sequence, and then cloned into pCAMBIA1300 (CAMBIA, http://www.cambia.org ). The resulting constructs of PMRG1::MRG1-GUS and PMRG2::MRG2-GUS were used to transform the mrg1 mrg2 plants. GUS activity was assayed by incubating plant tissues in GUS staining buffer [49] (link) for 3–6 hours at 37°C. Plant material was cleared in 70% ethanol, and observed directly under a dissecting microscope (MZ10F, Leica, Germany).
5
Visualization of Male Gametophyte Development
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Male gametophyte development was observed by both differential interference contrast (DIC) microscopy and inflorescence microscopy. For DIC microscopy, the pollens of different development stages were obtained and cleared by chloral hydrate solution (chloral hydrate: H2O: glycerol = 8: 2: 1) on slides. Cleared anthers were imaged using a BX63 microscope (Olympus) with DIC optics. For inflorescence microscopy, the samples were decoloured in 25% acetic acid 75% ethanol solution for three times and stained with 4′, 6-Diamidino-2-Phenylindole (DAPI), following the method described by Yang et al. (2009) and Dou et al. (2011) [54 (link), 55 (link)]. The nuclei of male gametophytes were then observed under Leica MZ10F and DM2500 microscopes.
6
Clonogenic Assay for Cell Proliferation
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Cells grown at 3% or 21% O2 were used at early passage (P2) and late passage (between P11 to P22). They were seeded at a clonal density of 5 cells/cm2 of a six-well plate in α-MEM supplemented with 10% fetal bovine serum (FBS) and incubated under a humidified atmosphere of 5% CO2 at 21% O2. After seven days, cells were fed with fresh medium for an additional period of seven days. At day 14, colony formation was obvious and the assay was stopped by fixation with cold absolute methanol for 10 min at 4 °C. Colonies were stained for 10 min in 0.5% crystal violet prepared in 25% methanol. After washing, colonies with more than 50 cells were scored by the use of a Leica MZ10F magnifying device. Results were analyzed by calculation of cloning efficiency E (E (%) = (Number of colonies/number of seeded cells) × 100).
7
GUS Staining in Arabidopsis Transgenic Lines
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GUS histochemical staining of Arabidopsis plants harboring AtGL2::GUS and AtDR5::GUS fusion constructs was performed as previously described (Prasinos et al., 2005 (link)) with minor modifications. Briefly, 5-day-old seedlings grown vertically on 1/2 MS agar medium supplemented with or without Cd (40 μM) and As(III) (10 μM) were immersed in GUS staining solution (50 mM NaPO4 buffer, pH 6.8, 0.5 M EDTA pH 8.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 20% triton X-100, and 2 mM X-gluc), and incubated overnight at 37°C. Photographs were taken with a microscope (Leica MZ10F, Germany) equipped with a digital camera (DFC450 C). GUS stained tissues represent the results of at least five independent seedlings.
8
Cacna1aa Knockdown Impacts Embryonic Development
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Mortality of eggs and larvae injected with Ctrl-MO and cacna1aa MO (individually and in combination) was assessed 4 h post-fertilization (hpf) and 24 hpf. Larvae were visually inspected for severe malformations (e.g., pericardial edema, body axis curvature, hemorrhage) from 1 to 5 dpf. For documentation, cacna1aa and Ctrl-MO morphants were photographed at 4 dpf, using a Leica MZ10F stereomicroscope equipped with a DFC310 FX digital camera.
9
ROS Visualization in Cadmium-stressed Roots
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Visualization of ROS in root was performed by using the CM-H2DCFDA [5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate] fluorescent probe (Molecular Probes1 ) as previously described (Wang et al., 2014 (link)), with some modifications. The roots of three-week-old plants grown in 1/2 MS medium supplemented with 0 and 50 μM of cadmium were harvested and briefly washed with deionized water. After immersing the roots in CM-H2DCFDA solution (10 μM) for 15 min in the dark, samples were rinsed with water and photographed using a fluorescence microscope (Leica, MZ10F, Germany) with 485 nm excitation and 510–530 nm emission filters under proper magnification (12.5-20X). Image J software (NIH, United States) was employed for quantification of the fluorescence signal. At least 10 plants were examined per line, and three independent biological experiments were performed.
10
Zebrafish Tail Fin Injury Assay
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Zebrafish were raised and maintained according to standard protocols (33 (link)) in U.K. Home Office-approved aquaria at the University of Sheffield Zebrafish Facility in the Bateson Centre. The transgenic Tg(mpx:GFP)i114 line (mpx:GFP), was used in both zebrafish experiments. Injury by tail fin transection was performed as previously described (34 (link)). Three days postfertilization (dpf) mpx:GFP zebrafish embryos were immersed in 0.02% 3‐amino benzoic acid ethyl ester (tricaine), then placed on masking tape on a Petri dish lid. The embryos were injured by complete tail fin transection at the caudal fin with a microscalpel (World Precision Instruments). Four hours following injury, zebrafish were visualized under a fluorescent dissecting microscope (Leica MZ10F) and embryos with 20–25 neutrophils at the injury site (posterior to the circulatory loop) were selected, transferred to a 96-well plate and polymersomes or their controls were added to the zebrafish media. Zebrafish were then washed and visualized using a Perkin-Elmer UltraVIEW VoX spinning disk confocal microscope or the number of neutrophils at the injury site was counted under a fluorescent dissecting microscope.
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