For live imaging of EB1-EGFP, cells were collected by standard trypsinization and 1.4 x 106 cells were plated on a collagen-coated 35 mm glass-bottom dish (MatTek Corporation). After recovering overnight, the spent media was replaced with fresh media containing diluted DMSO/nocodazole. Each dish was treated and imaged separately at room temperature using an Eclipse TE2000 confocal microscope (Nikon). After imaging up to 15 min, treated plates were incubated in 5% CO2 at 37°C for an additional 1.75 hours. Adhesion activation was then analyzed via brightfield microscopy with images collected using a IX-71 microscope (Olympus), the same as similar assays in this paper. Cells were then fixed in -20°C methanol for 10 min, washed twice with PBS, then blocked for 30 min in 5% milk/PBST before overnight incubation with mouse anti-α-tubulin DM1A monoclonal antibody (Pierce, #62204) [1:2,500] at 4°C. Cells were then washed twice with PBS before incubation with rabbit anti-mouse IgG (H+L) 546 [1:500] in the dark for 2 hours at room temperature. Cells were washed twice and left in a third wash of PBS for imaging on an Eclipse TE2000 confocal microscope (Nikon).
Eclipse te2000 confocal microscope
Manufactured by Nikon
Sourced in Japan
The Nikon Eclipse TE2000 is a confocal microscope designed for high-resolution imaging. It features a laser scanning system and advanced optics for capturing detailed, high-quality images of biological samples.
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Lab products found in correlation
Hoechst 33342, by Merck Group (6 mentions)
6 well plate, by Corning (4 mentions)
Laminin, by Merck Group (2 mentions)
Ix71 microscope, by Olympus (1 mentions)
35 mm collagen coated glass bottom dish, by MatTek (1 mentions)
Texas red phalloidin, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
E cadherin, by BD (1 mentions)
P smad1 5 9, by Cell Signaling Technology (1 mentions)
Draq5, by Biostatus (1 mentions)
Snai2, by Santa Cruz Biotechnology (1 mentions)
Cryostat, by Leica (1 mentions)
P36970, by Thermo Fisher Scientific (1 mentions)
Wga alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg alexa 488, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Alexa fluor goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Em ccd, by Nikon (1 mentions)
18 protocols using eclipse te2000 confocal microscope
1
Live Imaging of EB1-EGFP in Cells
2
Immunostaining analysis of EMT markers
3
Quantification of Viral Gene Expression
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Approximately 48 hours following CED of ZsGreen BPNs, mice were euthanized and transcardially perfused with 10 ml of 2% heparinized 0.9% saline, followed by 10 ml of tris-buffered saline with calcium chloride (0.1 g/liter). Brains were removed, rapidly frozen to −80°C, and cut into 100-μm sections using a cryostat (1905, Leica, Buffalo Grove, IL). Every other section within 2 to 3 mm of the injection site was collected on a slide and mounted with permanent mounting medium (P36970, Invitrogen, Carlsbad, USA). Sections were imaged using a Nikon Eclipse TE2000 confocal microscope (Nikon, Melville, NY) under ×4 magnification. Multiple images were taken and stitched together in montages to capture the entire injection site. Volume of transfected tumor tissue was quantified from these images using a MATLAB script similar to previous studies (32 (link)). Briefly, background fluorescence was subtracted and images were thresholded at 5% of the maximum intensity. The total volume of transgene expression was calculated by multiplying the area of distribution from each slice by the slice thickness and summing values for each slice.
4
FAP Immunofluorescent Staining in Cells
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Next, 3D immunofluorescent staining of FAP in cells of various lines was performed by using as primary antibodies FAP Antibody (F11-24): sc-65398 (Santa Cruz Biotechnology, Dallas, TX, USA) and as secondary antibodies conjugated with fluorescent dye–Goat anti-Mouse IgG Alexa488 (Thermo Fisher Scientific, Waltham, MA, USA). The membrane was stained with WGA Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA). Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) was used to visualize nuclei. Slides were analyzed using Eclipse TE2000 confocal microscope (Nikon, Minato-ku, Tokyo, Japan).
5
Immunofluorescence Imaging of Permeabilized Cells
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Fixed cells were permeabilized with 0.5% Triton X-100, 1% bovine serum albumin, 100 mM glycine, and 0.2 mg/ml EDTA in PBS on ice for 10 min. The cells were subsequently digested with RNase A in PBS-EDTA (5 mM) solution for 30 min at 37°C. Cells were blocked in 10% goat serum in PBS and 0.01% sodium azide for 1 h at 37°C or overnight at 4°C. For IF staining, cells were incubated with appropriate primary antibody diluted in blocking solution for 1 h at 37°C. After three washes using 0.05% Tween 20 in PBS, cells were incubated with a corresponding fluorescence-tagged secondary antibody [Alexa Fluor goat anti-mouse or Alexa Fluor goat anti-rabbit (Invitrogen)]. After three washes, cells were mounted with ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen). Stained cells were visualized and imaged using a Hamamatsu EM-CCD digital camera attached to the Nikon Eclipse TE2000 confocal microscope.
6
Localization of Nanoparticles in Cells
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For confocal microscopy, cells were grown overnight on sterile cover slips in 200 µl of a complete culture medium in 6-well plates (CoStar, Cambridge, MA, USA). NPs (50 µg/ml) were added and incubated with the cells for 24 h. Hoechst 33342 (Sigma-Aldrich), MitoTracker® Red, LysoTracker® Red DND-99, human transferrin-Alexa Fluor® 568 Conjugate, and Wheat Germ Agglutinin-Alexa Fluor® 555 Conjugate (all from Life Technologies, Waltham, MA, USA) used to localize NPs inside cells were added for the last 1 h of incubation. Before the analysis, extracellular FITC fluorescence was quenched with Trypan blue solution (0.1%) for 10 min as described earlier.19 (link) After that, cells were washed with a fresh medium, fixed with 1% paraformaldehyde, washed, and polymerized with Mowiol 4.88 medium (Calbiochem, Germany). Slides were analyzed using an Eclipse TE2000 confocal microscope (Nikon, Japan).
7
Immunohistochemical Analysis of Embryonic Patterning
8
Microscopic Analysis of Intestinal Pathology
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AC-NPs were overloaded onto a microscopic glass slide, air dried, covered with a cover glass in a polymerizing medium Mowiol 4.88 (Calbiochem), and analyzed using Eclipse TE2000 confocal microscope (Nikon).
For the analysis of intestinal pathology fractions of small intestine were excised 5 h post injection of the furano-allocolchicinoid preparations, washed out from feces, fixed with 4% paraformaldehyde, frozen in Tissue-Tek (Sakura, Alphen aan den Rijn, The Netherlands), and cryosectioned (ThermoScientific, Waltham, MA, USA). Control sections were stained with DAPI to visualize nuclei; experimental sections were additionally stained with phallaidin-Alexa488 (Life technologies, Waltham, MA, USA) staining actin microfilaments. Analysis was conducted using confocal microscope Nikon E2000 (Tokyo, Japan).
For the analysis of intestinal pathology fractions of small intestine were excised 5 h post injection of the furano-allocolchicinoid preparations, washed out from feces, fixed with 4% paraformaldehyde, frozen in Tissue-Tek (Sakura, Alphen aan den Rijn, The Netherlands), and cryosectioned (ThermoScientific, Waltham, MA, USA). Control sections were stained with DAPI to visualize nuclei; experimental sections were additionally stained with phallaidin-Alexa488 (Life technologies, Waltham, MA, USA) staining actin microfilaments. Analysis was conducted using confocal microscope Nikon E2000 (Tokyo, Japan).
9
Confocal Analysis of Compound 7 Effects
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For confocal analysis, cells were grown overnight on sterile cover slips inserted into 6-well plates (Costar, Washington, WA, USA) in 200 µL of complete culture medium. Compounds 7 were added in 10 µL and incubated with the cells overnight. LysoTrackRed, WGA-AlexaFluor555 (Applied Biosystems, Foster City, CA, USA), and Hoechst 33,342 (Sigma, Merck KGaA, Darmstadt, Germany) were added for 30 min. Cells were fixed with 1% paraformaldehyde, washed, and polymerized with Mowiol 4.88 medium (Calbiochem, Germany). Slides were analyzed using an Eclipse TE2000 confocal microscope (Nikon, Japan). ROS production was analyzed in the same way 2 h after the addition of a compound of type 7 .
10
Immunostaining Analysis of Cell Lines
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Immunostaining protocols are described in the Supplementary Experimental Procedures . WM samples were analyzed using the Zeiss LSM800 and Olympus FluoView FV10i confocal laser scanning microscopes. iPSCs and inducible-TAp73-Saos-2 samples were analyzed using a Zeiss LSM800 Confocal Laser Scanning Microscope and a Nikon Eclipse TE2000 Confocal Microscope. Confocal z-stack images were taken in all cases. Images were processed using ZEN blue software, FV10-ASW 2.1 viewer software, EZ-C1 software, and ImageJ software.
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