GFP and dsRed fluorescence images were collected using a Nikon Eclipse epifluorescence microscope and OpenLab software, and quantification was carried out using ImageJ. Averages of the mean fluorescence of the entire embryo were calculated to compare expression levels and backgrounds were subtracted using identically sized areas from an empty region of the slide. Fluorescence intensities provided in the figures are in arbitrary units. P-values were determined using a two-tailed Student’s t-test.
Eclipse epifluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse epifluorescence microscope is a high-performance laboratory instrument designed for fluorescence imaging applications. It offers a range of features and capabilities to facilitate advanced microscopy research and analysis.
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Lab products found in correlation
Cryostat, by Thermo Fisher Scientific (3 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (3 mentions)
Mvx10 macroview microscope, by Olympus (2 mentions)
Ix83 p2zf inverted microscope, by Olympus (2 mentions)
Cellsens software version 3, by Olympus (2 mentions)
Live dead sperm viability kit, by Thermo Fisher Scientific (2 mentions)
Anti rhodopsin, by Abcam (2 mentions)
Donkey anti rabbit or mouse alexa fluor 488 or 568, by Thermo Fisher Scientific (2 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (2 mentions)
Syto 16 green fluorescent nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Fitc conjugated goat anti mouse igg, by LGC (1 mentions)
Columbus software, by PerkinElmer (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Tissue freezing medium, by Electron Microscopy Sciences (1 mentions)
Anti nitrotyrosine, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Fv1000 microscope, by Olympus (1 mentions)
19 protocols using eclipse epifluorescence microscope
1
Quantitative Analysis of Fluorescence Images
2
Immunofluorescence Analysis of Mecp2 and Ki67
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To evaluate the expression of Mecp2 or Ki67 during the phase of liver generation, primary antibodies, including anti-Alb, anti-Mecp2, or anti-Ki67, were added to sections for 12 hr at 4°C. Sections were washed three times in PBS, followed by application of secondary antibody goat anti-mouse Alexa Fluor 488 or donkey anti-rabbit Alexa Fluor 594 at a 1:200 dilution for 1 hr at room temperature. Nuclei were counterstained using DAPI. Following a wash in PBS, tissues were mounted with 50% glycerol and viewed on a Nikon (Tokyo, JP) Eclipse epi-fluorescence microscope.
IF procedures were performed as follows: G0 and cell cycle re-entry NIH3T3 cells were fixed with 4% paraformaldehyde and then washed twice with PBS. Cells were then permeabilized with 0.2% Triton X-100 at 4°C for 15 min and subsequently blocked with 1% bovine serum albumin (BSA) for 60 min at room temperature. Primary antibodies included anti-Mecp2, anti-Nedd4, and anti-Ki67, which were incubated overnight at 4°C in the presence of 1% BSA. The cells were then visualized using secondary antibody conjugated to Alexa Fluor-488 or Alexa Fluor-594 as described above.
IF procedures were performed as follows: G0 and cell cycle re-entry NIH3T3 cells were fixed with 4% paraformaldehyde and then washed twice with PBS. Cells were then permeabilized with 0.2% Triton X-100 at 4°C for 15 min and subsequently blocked with 1% bovine serum albumin (BSA) for 60 min at room temperature. Primary antibodies included anti-Mecp2, anti-Nedd4, and anti-Ki67, which were incubated overnight at 4°C in the presence of 1% BSA. The cells were then visualized using secondary antibody conjugated to Alexa Fluor-488 or Alexa Fluor-594 as described above.
3
Fluorescence Imaging of Auxin-Treated Samples
4
Cell-ECM Adhesion Quantification by Flow
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Cell-ECM attachment was measured by subjecting adhering cells to flow-induced shear stress as previously described [1 (link)]. Briefly, DMSO or PD0325901-treated GBM cells were plated at a concentration of 5x104 cells/ml onto 6-well polyethylene terephthalate cell culture inserts (Franklin Lakes, NJ) for 2 h and were then inverted into complete medium and incubated overnight. Inserts were then loaded into custom-designed flow chambers and subjected to 30 dynes/cm of shear stress for 3 h, whereupon inserts were washed in PBS and immersed in SYTO 16 green fluorescent nucleic acid stain (Life Technologies, Carlsbad, CA). Cells seeded onto inserts but not subjected to flow were used as growth rate controls. A Nikon Eclipse epifluorescence microscope was used to capture nine low magnification fields/insert and nuclei were counted in ImageJ. The average number of attached cells was then expressed as a percentage of the no-flow controls.
5
Indirect immunofluorescence for ANAs
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To obtain serum, before sacrificing the mice we bled each one by puncturing of the submandibular vein [40 ]. Following that, anti-nuclear autoantibodies (ANAs) were determined by indirect immunofluorescence assay. Each serum sample at 1:50 dilution was added to Hep-20 slides (EUROIMMUN, Lübeck, Germany). Slides were incubated at room temperature in a moist chamber for 30 minutes. Subsequently, they were stained with FITC-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories, Maryland, USA) in a dilution of 1:500 (1mg/mL) for 30 minutes. Slides were visualized using an Eclipse epifluorescence microscope (Nikon, Tokyo, Japan) and images were analyzed with ImageJ software (NIH, Bethesda, Maryland).
6
Cloning and Expression of GFP-PFT Fusion
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RNA was extracted from the wheat cultivar Sumai 3 and full-length PFT was amplified and cloned into entry vector pDONR221, using Gateway BP clonase reaction. PFT was fused with enhanced GFP at the N terminus and was cloned into plant expression destination vector pK7FWG2, using LR clonase reaction (Karimi et al. 2002) (link). The expression of the GFP:PFT fusion construct was driven by the 35S CaMV promoter. After confirmation with Sanger sequencing, the GFP:PFT fusion construct was electroporated in Agrobacterium tumefaciens GV3101, which was used for transforming Arabidopsis thaliana ecotype Ler, using the floral dip method (Clough and Bent 1998). T 0 seeds were plated on Murashige-Skoog media with kanamycin (50 mg/liter), and the resistant T 1 plants were further screened for GFP signal, using a Nikon Eclipse epifluorescence microscope. T 1 plants were grown to produce T 2 seeds, which were further selected for homozygous individuals. Two independent homozygous T 2 families were used for disease assays. Further, four T 3 plant families were further selected on kanamycin-supplemented media and for GFP fluorescence and were subsequently used for disease assays.
7
Intestinal Microbiome Imaging Protocol
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A segment of intestinal tissue extending from the distal ileum to proximal colon was harvested from mice post mortem, after behavior testing was complete. The tissue was fixed in Carnoy’s fixative (American MasterTech Scientific, Inc., Lodi, CA, USA) at room temperature for 24 hours, then incubated in dry methanol prior to processing and embedding. 4 μm sections were mounted on glass slides, baked at 60°C for 1 hour, then de-paraffinized with xylene and dehydrated in series from 50% to 100% ethanol. The probes used were a previously validated [41 (link)], 5’ Texas Red-labelled Bifidobacterium genus specific probe (Bif164; 5’-CATCCGGCATTACCACCC-3’ ) or a 5’ Alexafluor 488-labelled universal bacterial probe (Uni519; 5’-GTATTACCGCGGCTGCTG-3’ ) targeted to the 164–181 bp or 519–536 bp regions of 16S rRNA, respectively (Integrated DNA Technologies, Coralville, IA, USA). The probe was hybridized to the samples by adding 15 μL of [2 μM] probe to each slide and placing in a 45°C hybridization chamber for 45 minutes. Nuclei were labeled with DAPI. Images were acquired using a Nikon Eclipse epifluorescence microscope. Representative images are shown in Fig 2 , although intestinal sections from mice in each cage (total 5–6 per group) were utilized to confirm bacterial colonization.
8
Fluorescence Microscopy of Auxin Treatments
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For the auxin treatment experiments in Figure S3, fluorescence images were obtained using an Olympus IX83 P2ZF inverted microscope. Z-stacks were captured with a step size of 0.2 µm, using a 100´ silicon oil immersion objective (1.35 N.A.). Images in Figures S5 andS6 were taken with an Olympus MVX10 MacroView microscope using a 2´ objective (0.5 N.A.). The rest of the DIC images and fluorescence images were obtained using a Nikon Eclipse epifluorescence microscope using 10´ (0.25 N.A.) and 40´ (0.75 N.A.) objectives. Images were acquired using Olympus cellSens software version 3.1. Before imaging, animals were mounted on 3% agarose pads and immobilized using 10 mM levamisole.
9
Sperm Viability Evaluation Using Fluorescence
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To evaluate a potential negative effect of viral suspension on sperm cells, viability of spermatozoa was analyzed before and after the above described incubation with viral suspension. Briefly, 25 µL of semen were incubated with 2 µL of a 300 µM Propidium Iodide (PI) stock solution and 2 µL of a 10 µM SYBR-14 stock solution (both obtained from the Live/dead sperm viability kit; Invitrogen, Eugene, OR, USA), for 5 min at 37°C in the dark. After incubation, 10 µL of sperm suspensions were analyzed with a Nikon Eclipse epifluorescence microscope using a double-band-pass filter for green and red fluorescence. The spermatozoa with green or red fluorescence on the head were considered live or dead, respectively. At least 200 spermatozoa were scored for each sample.
10
Sperm Viability Evaluation via Fluorescence
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Sperm viability was evaluated by incubating 25 µL of semen with 2 µL of a 300 µM Propidium Iodide (PI) stock solution and 2 µL of a 10 µM SYBR-14 stock solution (LIVE/DEAD®Sperm Viability kit, Molecular Probes, Invitrogen), for 5 min at 37°C in the dark. After incubation, 10 µL of sperm suspensions were analyzed with a Nikon Eclipse epifluorescence microscope using a double-bandpass filter for green and red fluorescence. The spermatozoa with green or red fluorescence on the head were considered live or dead, respectively (see supplementary file, panel A). At least 200 cells were counted in each analysis.
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