Flowers and floral organs were photographed with a MZ16FA stereoscopic microscope (Leica, Wetzlar, Germany). Mature pollen grains were stained with Alexander’s solution (95% ethanol, 10 mL; 1% acid fuchsin, 5 mL; 1% orange G, 0.5 mL; glacial acetic acid, 2 mL; glycerol, 25 mL; phenol, 5g; distilled water, 50 mL) overnight [92 (link)], 1 μg·L−1 DAPI solution for 5 min [93 (link)] and calcofluor white solution for 5 min [94 ], respectively. Images of stained pollen grains were taken using an ECLIPSE 90i fluorescent microscope (Nikon, Tokyo, Japan). Pollen germination in vitro was performed in a medium containing 15% sucrose, 0.4 mmol·L−1 HBO3, 0.4 mmol·L−1 Ca(NO3)2, 0.1% agar, and the pH adjusted to 5.8. The pollen grains were grown at 22 °C with 100% relative humidity in the dark. Pollen tubes were photographed with an ECLIPSE 90i fluorescent microscope (Nikon, Tokyo, Japan). For scanning electron microscopic examination, fresh anthers and pollen grains were coated with gold–palladium in a Model IB5 ion coater (Eiko Engineering Company, Nagoya, Japan) and observed under a Model TM-1000 microscope (Hitachi, Tokyo, Japan).
Eclipse 90i fluorescent microscope
Manufactured by Nikon
Sourced in Japan, United States
The Nikon Eclipse 90i is a fluorescent microscope designed for advanced imaging and analysis. It features a high-performance optical system, a wide range of illumination options, and a versatile modular design to accommodate various research and laboratory applications.
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Lab products found in correlation
Histology fish accessory kit, by Agilent Technologies (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Mz16fa stereoscopic microscope, by Leica (1 mentions)
Dq gelatin, by Thermo Fisher Scientific (1 mentions)
Dako hybridizer, by Agilent Technologies (1 mentions)
Opti mem 1 reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Mowiol, by Merck Group (1 mentions)
Autoquant x3, by Media Cybernetics (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Fluoromount g, by Electron Microscopy Sciences (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti keratin 14, by Fortrea (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Mouse anti p63, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti loricrin, by Fortrea (1 mentions)
Anti insulin, by Agilent Technologies (1 mentions)
Dapi nuclear stain, by Thermo Fisher Scientific (1 mentions)
Anti β galactosidase, by Abcam (1 mentions)
14 protocols using eclipse 90i fluorescent microscope
1
Comprehensive Pollen Visualization and Germination
2
In-situ Zymography of Gelatinase Activity
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Mice received either sham or injury and were treated with vehicle or p-OH SB-3CT (n = 4/group), at the same dosage and timing as for all other studies (S1 Fig ). One hour after the last injection, brains were collected and embedded in optimal cutting temperature medium (OCT, Tissue-Tek, CA). The tissue blocks were cryosectioned at 10μm thickness and subjected to in-situ zymography, as described by Oh et al. [30 (link)]. Briefly, the sectioned tissues were stained with DQ-gelatin (Molecular Probes, Inc., OR, USA) to detect gelatinase activity, followed by incubation with DAPI solution to detect nuclei. The images for in-situ zymography were obtained using same exposure setting for all sections examined by confocal microscopy (Nikon Eclipse 90i Fluorescent Microscope (Nikon Instruments Inc., Melville, NY). No image processing was performed to adjust signal intensities of DQ-gelatin.
3
TMPRSS2-ERG Fusion Assessment by FISH
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The TMPRSS2-ERG status of samples in the validation cohort was determined by using a break-apart assay with a triple-labeled color commercial probe (Kreatech Diagnostics, The Netherlands). The probe detects the deletion between TMPRSS2 and ERG at 21q22. The FISH assay was carried out on 4 μm formalin-fixed, paraffin-embedded tissue sections after deparaffinization which were then pretreated using a commercial tissue section kit for paraffin-embedded tissue (Histology FISH Accessory Kit, Dako). The probe mix was applied and denatured at 80°C for 5 minutes before hybridization at 37°C overnight using a Dako hybridizer. The slides were counterstained with DAPI (4′,6-diamidino-2-phenylindole) from the Histology FISH Accessory Kit. Results were visualized using a 100x oil immersion objective on a Nikon Eclipse 90i fluorescent microscope (Nikon Corp., Japan) equipped with appropriate filters. For each sample, 25 non-overlapping nuclei in cancer areas were evaluated for deletion of the TMPRSS2 (21q22) gene region associated with TMPRSS2-ERG. In order to compensate for nuclear truncation, the cut-off level for an informative result was defined as loss of the TMPRSS2 (21q22) gene region at least 80% of tumor cell nuclei.
4
In Vivo Fibrinogen Biodistribution
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Unmodified and PEGylated fibrinogen solutions (35 µl, containing Alexa Fluor™ 647 labelled fibrinogen at 1.5% m/m unlabelled fibrinogen) were prepared and injected into the tibialis anterior (TA) muscle of BALB/c mice 5 min after mixing. The mice were euthanized at 30 min, 2-, 4- and 7-days post injection. Both TA muscles per mouse were utilized and two mice were used per time point. TA muscle samples were dissected out and immediately fixed in 10% formalin (v/v). After fixation, tissue samples were processed for wax embedding and then sectioned on a microtome. Stitched fluorescent micrographs were captured on a Nikon Eclipse 90i fluorescent microscope (Nikon, Japan) with the same exposure settings used for all. Alexa Fluor™ 647 labelled fibrinogen was quantified using Visiopharm image analysis software (Visiopharm, Denmark).
5
Transfection of Alpha-Synuclein Variants in Cholinergic Cells
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The transient transfection of plasmids in RA-differentiated cholinergic cells was accomplished by utilizing Lipofectamine 3000 reagents (Thermo Fisher Scientific, Waltham, MA, USA; Cat. #: L3000001) to ensure maximal gene expression with minimal cellular toxicity. Briefly, 0.1 μg of pEGFP-C1, 0.1 μg of pcDNA6 wild-type α-syn, or 0.1 μg of pcDNA6 E46K mutant α-syn plasmids were diluted in 5 μL of Opti-MEM I reduced serum medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat. #: 31985070) and 0.2 μL of P3000 reagent. Following incubation for 5 min at room temperature (RT), 0.3 µL of Lipofectamine 3000 reagent was diluted in 5 μL of Opti-MEM I reduced serum medium, added into the above-diluted Opti-MEM-DNA solution, and incubated for an additional 15 min to form DNA-Lipofectamine 3000 complexes. The complex mixtures were then added dropwise into each well of a 96-well plate and incubated in a humidified 5% CO2 incubator for two to four days. Finally, the evaluation of transfection efficiency by visualization of GFP expression in RA-differentiated cholinergic cells was done by counting the number of GFP-positive cells using a Neubauer hemocytometer under a Nikon Eclipse 90i fluorescent microscope (Nikon, Melville, NY, USA).
6
Transient Transfection of Plasmid DNA in RA-Differentiated Cholinergic Neurons
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The transient transfection of plasmid DNA in RA-differentiated cholinergic neurons was accomplished by employing Lipofectamine 3000 reagents (Thermo Fisher Scientific, Waltham, MA, USA; Cat. #: L3000001). Briefly, 2.5 μg of pEGFP-C1, 2.5 μg of pcDNA6 wild-type α-syn, or 2.5 μg of pcDNA6 E46K mutant α-syn plasmids were diluted in 125 μL of Opti-MEM serum-reduced medium (Thermo Fisher Scientific, Waltham, MA, USA; Cat. #: 31985070) and 5 μL of P3000 reagent, respectively. Succeeding a 5 min incubation step at room temperature (RT), 7.5 µL of Lipofectamine 3000 reagent was diluted in 125 μL of Opti-MEM serum-reduced medium, added into the diluted Opti-MEM-DNA solution, and incubated for an additional 15 min to assemble DNA-Lipofectamine 3000 lipoplexes. The complex concoctions were thereafter added dropwise into each well of a 6-well plate and re-incubated in a humidified 5% CO2 incubator for 2–4 days. The estimation of transfection efficiency in RA-differentiated cholinergic neurons was achieved by enumerating the amount of GFP-positive neurons under a Nikon Eclipse 90i fluorescent microscope (Nikon, Melville, NY, USA).
7
Subcellular Localization of BoPG Proteins
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We selected four BoPGs which had higher expression levels in inflorescences, to investigate their subcellular localizations by generating C-terminal green fluorescent protein (GFP) fusions. Based on their CDS provided by the Brassica database, we designed four specific primers (Supplementary Materials Table S8 ) for nucleotide fragment amplification. The PCR amplification reaction was carried out by using the high-fidelity enzyme KOD-plus-Neo, in accordance with the manufacturer’s instruction. The resulting fragment was cloned into the fusion expression vector pFGC–GFP under the control of the constitutive CaMV35S promoter, to create the fusion vector. Colonies containing the appropriate insert were identified by sequencing. The fusion vector was then transiently transformed into onion epidermal cells by particle bombardment. The fluorescence signal was analyzed after 16 h of incubation with the Nikon Eclipse 90i Fluorescent Microscope (ECLIPSE 90i; Nikon). The onion epidermal cells were plasmolyzed by infiltrating 0.3 g·mL−1 sucrose.
8
Immunostaining and In Situ Hybridization of Mouse Embryos
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Section staining and microscopy For immunostaining of spinal cord cross-sections, mouse embryos were collected at E11.5, fixed in 4% PFA for 1 hour at 4 C or at room temperature, transferred to 10-30% sucrose in PBS at 4 C overnight, then embedded in gelatine-sucrose or OCT and frozen. 16-20 mm sections were cut. Samples were then blocked with PBS + 3% or 10% donkey serum + 0.1% Triton X-100 in PBS pH 7.4 for 1 hour at room temperature. The blocking solution was replaced with the primary antibody diluted in 1% PHT (1% heat-inactivated donkey serum, 0.1% Triton X-100 in PBS pH 7.4) and incubated overnight at 4 C. The secondary antibody was diluted in 1% PHT and incubated for 1 hour at room temperature. Probes for Netrin-1 in situ were generated targeting exon 2 of the mouse netrin-1 gene to validate the conditional allele removal (Brunet et al., 2014) . In situ hybridization was performed as described (Marillat et al., 2002) . Coverslips were mounted in Fluoromount G (Electron Microscopy Sciences) or in Mowiol (Sigma) and imaged with a Nikon Eclipse 90i fluorescent microscope with a 10X objective coupled to a Nikon QiMc camera (Nikon, Japan) or a Leica SP8 confocal microscope with 20X objective (Leica, Germany). Image stacks were deconvolved with AutoQuant X3 software (Media Cybernetics) to enhance the spatial signal allocation.
9
Immunofluorescent Detection of Epidermal Markers
10
Dual Fluorescence Staining of Pancreas Islet Cells
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Pancreas tissue was fixed in buffered 10% formalin, processed to wax blocks and sectioned onto slides (4 μm). Dual fluorescence staining was performed to determine the islet cell type in which β-galactosidase/GPR120 was expressed. Anti-insulin (1:500; Dako, Cambridge, UK), anti-glucagon (1:200; Abcam, Cambridge, UK) and anti-somatostatin (1:100; Millipore, Watford, UK) were detected using Alexa Fluor-488 conjugated anti-goat antibody (Abcam) and anti-β-galactosidase (1:1200; Abcam) with Alexa Fluor-568 conjugated anti-rabbit antibody. Citrate buffer (pH 6) was used for antigen retrieval in the case of β-galactosidase and somatostatin. Briefly, tissue sections were fixed, de-waxed and rehydrated prior to antigen retrieval (heating in a microwave at full power for 20 min followed by cooling to room temperature). The sections were blocked before the addition of primary antibodies and washed prior to the addition of secondary antibodies and DAPI nuclear stain (1:500; Thermo Scientific). Slides were mounted in Hardset Vectashield Mounting medium (Vector Laboratories, Peterborough, UK) and analysed on a Nikon Eclipse 90i Fluorescent Microscope.
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