Embryos were fixed in 4% PFA/PBS for 30 min and washed three times for 10 min in PBS-T (0.1% Tween). Specimens were then stained at 37°C in a solution containing 5 mM potassium hexacyanoferrate (III), 5 mM potassium hexacyanoferrate (II) trihydrate, 2 mM magnesium chloride, 0.01% sodium deoxycholate, 0.02% NP-40, and 1 mg/mL X-gal solution in PBS. After proper staining was achieved, the specimens were washed three times for 15 min in PBS-T, fixed again in 4% PFA/PBS for 30 min, and washed again. Images were taken with a Leica MZFLIII microscope. WISH was performed as described in Woltering et al. (2009) (link). Images were taken with a Leica MZFLIII microscope.
Mzfliii microscope
Manufactured by Leica
Sourced in Germany
The MZFLIII is a stereomicroscope designed for a wide range of applications. It features high-performance optics, a flexible stand, and a variety of accessories to support various tasks. The MZFLIII provides clear, high-contrast images and a large working distance to accommodate different specimen sizes and types.
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Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Cryostat, by Leica (4 mentions)
Dfc320, by Leica (3 mentions)
Paraplast plus, by Merck Group (3 mentions)
Mmessage mmachine t7 kit, by Thermo Fisher Scientific (2 mentions)
Dfc300fx camera, by Leica (2 mentions)
Rm2265 microtome, by Leica (2 mentions)
Firecam software, by Leica (2 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Balb c, by Charles River Laboratories (1 mentions)
Examiner lsm880, by Zeiss (1 mentions)
Dfc300fx, by Leica (1 mentions)
Gateway recombination cloning technology, by Thermo Fisher Scientific (1 mentions)
Dfc450c, by Leica (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Renilla luciferase control reporter vector, by Promega (1 mentions)
Pgl3 promoter vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
59 protocols using mzfliii microscope
1
Whole-Mount X-Gal Staining of Embryos
2
Brain Slice Preparation and Staining
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Rats from Experiment 2 were overdosed on pentobarbital (0.5 ml, i.p.) and intracardially perfused with physiological saline and 10% formalin. Brains were extracted and stored in 10% formalin for twenty-four hrs then switched to a sucrose-formalin solution until sectioning. Brains were flash frozen and sectioned at 40 μm on a cryostat. Every third slice was wet-mounted to a gel-subbed microscope slide. Sections were stained with 0.25% thionin. Photomicrographs of brain slices were generated for each rat using a Leica MZFLIII microscope. Only rats with injector tips localized within the BLA (bilaterally) were included in the final analyses.
3
Cryosectioning and Cresyl Violet Staining
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Dissected brains were infiltrated with 30% sucrose in PBS, embedded in optimum cutting temperature (OCT) compound, and sectioned at 20 μm on a cryostat. Every 10th section was placed on a slide and stained in 0.1% cresyl violet solution for 10 min, rinsed quickly in PBS, dehydrated in 95% ethanol, and left in xylene before being coverslipped with Permount (Fisher Scientific). Brightfield images were taken using a Leica MZFLIII microscope with a Leica DFC425 camera and LAS V3.8 software.
4
Whole-mount in situ Hybridization of Brachyury and Cer1
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Whole-mount in situ hybridization of Brachyury and Cer1 markers was performed using standard procedures (Eggenschwiler and Anderson 2000 (link)). A 282-bp β-Pix RNA probe was generated using the following primers: 5′-AAACCTTTCAGCTCAGTGTCAAG-3′ and 5′-AGCTTTGTGATTGTCATTCCTGT-3′. Embryos were fixed in 4% paraformaldehyde and processed using whole-mount in situ hybridization. LacZ staining was performed as described previously (Migeotte et al. 2010 (link)). After in situ hybridization or LacZ staining, whole-mount embryos were imaged using a Zeiss Axiocam HRC digital camera or Canon EOS camera on a Leica MZFLIII microscope. LacZ in histological sections was imaged using a 10× Plan-Neofluar NA 0.5 Zeiss Axio microscope equipped with a color Axiocam camera.
5
Mammary Tumor Implantation and Imaging in Mice
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Balb/C (for 4T1 tumors) and Balb/C-nu/nu (CAnN.Cg-Foxn1nu/Crl; for MMT tumors) female mice (6-8 weeks old) were obtained from Charles River Laboratories. Animal procedures were approved by the Animal Ethics Committee of Radboud University, Nijmegen (RU-DEC 2012-129) and performed according to the guidelines of the Dutch Act on Animal Experimentation and the European FELASA protocol.
For implantation of the mammary window, the 4th mammary fat pad in anesthetized mice (1-2% isoflurane in oxygen) was exposed by surgical incision using microsurgery under a Leica MZFLIII microscope. A superficial microchannel was created within the fat pad using a 30 G syringe needle (BD Medical) into which an individual spheroid in a microdrop (2 µl in PBS) was implanted using a gel-loading pipet tip (0.3 mm inner diameter, BIOplastics). After implantation of up to 10 spheroids in the same tissue area, a mammary imaging window was inserted, affixed to the skin by gentle tightening of a purse-string suture around the skin rim, and closed with a cover glass and spring ring, as described (Alieva et al., 2014 (link)). Multifocal tumor development was monitored by whole-body fluorescence imaging (FluorVivo100, INDEC BioSystems) and quantified as total tumor area in the mammary window from 2D projections of the DsRed2 and H2B-eGFP signal.
For implantation of the mammary window, the 4th mammary fat pad in anesthetized mice (1-2% isoflurane in oxygen) was exposed by surgical incision using microsurgery under a Leica MZFLIII microscope. A superficial microchannel was created within the fat pad using a 30 G syringe needle (BD Medical) into which an individual spheroid in a microdrop (2 µl in PBS) was implanted using a gel-loading pipet tip (0.3 mm inner diameter, BIOplastics). After implantation of up to 10 spheroids in the same tissue area, a mammary imaging window was inserted, affixed to the skin by gentle tightening of a purse-string suture around the skin rim, and closed with a cover glass and spring ring, as described (Alieva et al., 2014 (link)). Multifocal tumor development was monitored by whole-body fluorescence imaging (FluorVivo100, INDEC BioSystems) and quantified as total tumor area in the mammary window from 2D projections of the DsRed2 and H2B-eGFP signal.
6
Fluorescent Imaging of Embryos
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Fluorescent stereoscope images were taken on a Leica MZFLIII microscope with an IDS UI‐1240SE camera.
Wholemount confocal fluorescent images were captured on a Zeiss Examiner LSM880 confocal using a 20x/NA1.0 or 10x/NA0.5 Plan Apochromat dipping objectives as previously described (Butler et al.,2019 ; Galea et al., 2017 ). Embryos were typically imaged with X/Y pixel sizes of 0.59 μm (speed = 8, bidirectional imaging, 1,024 × 1,024 pixels). Images were processed with Zen2.3 software and visualized as maximum projections in Fiji.
Wholemount confocal fluorescent images were captured on a Zeiss Examiner LSM880 confocal using a 20x/NA1.0 or 10x/NA0.5 Plan Apochromat dipping objectives as previously described (Butler et al.,
7
Tracing Adipocyte Lineage in Mice
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Male congenic C57BL/6J mice hemizygous for a Fatty Acid Binding Protein 4 (Fabp4)-Cre transgene (35 (link)) were mated to homozygous R26R-EYFP females (36 (link)). The +/Fabp4-Cre;+/R26R-EYFP and +/+;+/R26R-EYFP (control) male offspring from these matings were genotyped as previously described (30 (link)). Freshly dissected organs were visualized with a Leica MZFLIII microscope (Wetzlar, Germany) and an epifluorescent yellow fluorescent protein (YFP) filter. Photographs were taken with a CoolSNAP camera and PMCapture Pro 6.0 software (Photometrics, Tucson, AZ, USA).
8
Quantifying Zebrafish Thymopoiesis via rag1/gh Ratio
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Thymic rag1 gene expression is a marker of ongoing assembly of T cell receptor genes. Thus, the intensity of the RNA in situ signal correlates with the number of differentiating T cells, which we consider to be a measure of T cell development. To provide an internal control (technical, with respect to the hybridization process as such, and biological, with respect to the tissue specificity of the observed genetic effects), we used a probe specific for the growth hormone (gh) gene, which marks a subset of cells in the hypophysis. Determination of rag1/gh ratios was carried out as follows. After RNA in situ hybridization with rag1 and gh probes, ventral images of 4 to 5 dpf zebrafish larvae were obtained with a Leica MZFLIII microscope and a Leica DFC300FX digital camera, essentially generating a 2D projection of the 3D structure. The areas of rag1 and gh signals were measured using ImageJ, and the ratio of average of the rag1-positive area vs. gh-positive area was calculated as a measure of thymopoietic activity. After photographic documentation of the RNA in situ hybridization signal, larvae were processed for genomic DNA extraction for subsequent genotyping.
9
Enhancer Identification using ZED Reporter
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Candidate enhancer sequences of different alleles with closely flanking sequences were constructed into ZED Vector (Bessa et al. 2009 (link)) using Gateway Recombination Cloning Technology (Thermo Fisher Scientific). T7-Transposase (Khattak et al. 2014 (link)) (Addgene) was transcribed using mMESSAGE mMACHINE T7 kit (Life Technologies), according to the manufacturer’s instructions. A final concentration of 40 ng/µl ZED constructs, 50 ng/µl transposase mRNA, and 0.05% phenol red were coinjected into one-cell stage embryos. The embryos were imaged for GFP and internal control red fluorescent protein (RFP) expression at different time points using a Leica MZFLIII microscope. The elements were considered as candidate enhancers if there were more than 20% of injected embryos showing consistent expression pattern of GFP at the presence of RFP (Bessa et al. 2009 (link); Sharma et al. 2015 (link)).
10
Imaging Techniques for Zebrafish Embryogenesis
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Live embryo and in situ hybridization images were captured at 4× or 10× magnifications using a ProgressC14 digital camera (Jenoptik) on a Leica MZFLIII microscope. Brightfield video microscopy of Kupffer’s vesicle and embryonic kidneys was performed on an inverted Leica SP5 spectral confocal microscope. Live embryos were mounted in 2% low-melt agarose in glass-bottom tissue culture dishes and illuminated with 561 nm wavelength light. Recordings were captured at 170 frames/s using a 63× glycerin immersion objective and beat frequency was analyzed as described.44 (link) Histology of zebrafish embryos was performed as described.45 (link)
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