Samples were visualized using an Olympus IX81 wide-field microscope, Hamamatsu C4742-95 digital camera, and Slidebook software; 100× objective (UPIanFI, 1.30 NA Oil) or Lionheart FX: Automated Microscope and Gen5 software; Olympus 60X Universal Plan Fluorite Dry Objective, 0.9 NA. Images were processed and analyzed using FIJI image analysis software. Any images appearing in the same panel of a figure were processed identically including objectives, acquisition settings, cropping, brightness adjustments, and any other processing settings.
Ix81 widefield microscope
Manufactured by Hamamatsu Photonics
The IX81 widefield microscope is a high-performance microscope system designed for a variety of imaging applications. It features a modular design that allows for customization to meet specific research needs. The IX81 provides a stable and versatile platform for a range of imaging techniques, including brightfield, fluorescence, and phase contrast microscopy.
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Lab products found in correlation
C9100 13 em ccd camera, by Hamamatsu Photonics (2 mentions)
Orca er ccd camera, by Hamamatsu Photonics (1 mentions)
Metamorph software, by GE Healthcare (1 mentions)
U mgfphq, by Olympus (1 mentions)
Ab84422, by Abcam (1 mentions)
Ab51081, by Abcam (1 mentions)
Orca r2 cooled ccd camera, by Hamamatsu Photonics (1 mentions)
Sc 8418, by Santa Cruz Biotechnology (1 mentions)
Np 40, by Merck Group (1 mentions)
5 protocols using ix81 widefield microscope
1
Wide-field Microscopy and Image Analysis
2
Widefield Imaging of Fluorescent Proteins
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Widefield micrographs were obtained on an Olympus IX81 widefield microscope equipped with a 100×/NA1.45 objective and an ORCA-ER CCD camera (Hamamatsu), using an X-CITE 120 PC (EXFO) metal halide lamp as the illumination source. The excitation and emission light when imaging EGFP- and mCherry-tagged proteins were filtered through the U-MGFPHQ and U-MRFPHQ filter sets (Olympus). The 3D stacks were acquired with 0.2 μm vertical spacing. The microscope was controlled using the MetaMorph software (Molecular Dynamics).
3
Multicolor Immunofluorescence Staining Protocol
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Cells were fixed for 5 min in 3.7% paraformaldehyde (PFA) (Sigma-Aldrich) followed by 2 min permeabilization with 0.5% NP-40 (EMD Millipore). The coverslips were incubated with blocking solution (1% bovine serum albumin) for 1 h at RT. Primary antibodies used for immune staining were diluted in blocking solution and applied to the coverslips for 1 h at RT; this was followed by three short rinses in PBS. Primary antibodies were as follows: anti-Matr3C (ab84422; Abcam), anti-Matr3N (ab51081; Abcam), and mouse anti-S6K used to detected Hedls (sc-8418; Santa Cruz) at 1:200; rabbit anti-Rck/p54 (A300-461A; Bethyl Laboratories, Montgomery, TX) and mouse anti-PABP-1 (10E10, sc-32318; Santa Cruz) at 1:200. Next the coverslips were incubated with secondary antibodies, diluted 1:1000 in blocking buffer, and conjugated to Alexa Fluor 405, 488, or 546 (Invitrogen, Carlsbad, CA) for 1 h at RT. The coverslips were next stained with 4′,6-diamidino-2-phenylindole and rinsed with PBS before being mounted onto slides using Mowiol mounting media. Images were acquired at RT using a using an Olympus IX81 wide-field microscope with a 40× air objective lens attached to a Hamamatsu Orca-R2 cooled CCD camera. Pearson’s colocalization coefficients were determined using the Coloc2 plug-in in imageJ.
4
Live tracking of Gata6 reporter expression
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To track Gata6 reporter expression in live cells, PrE-like differentiation was induced by a 6 h pulse of doxycycline-treatment in serum-containing medium as described in (Schröter et al., 2015 (link)). Then, 16 h after doxycycline removal, cells were either switched directly to N2B27 medium lacking Phenol Red or trypsinized, sorted for reporter expression and seeded on fibronectin-coated imaging dishes (ibidi µ-slides). Time-lapse imaging was started within 2 h after sorting on an Olympus IX81 widefield microscope equipped with LED illumination (pE4000, CoolLED) and a Hamamatsu c9100-13 EMCCD camera. Hardware was controlled by MicroManager software (Edelstein et al., 2001 ). Time-lapse movies were acquired using a 40× oil immersion lens (NA 1.2), with 10 min time intervals.
Cell tracking was carried out with TrackMate (ImageJ) (Tinevez et al., 2017 (link)) based on the constitutively expressed H2B-Cerulean nuclear marker. Fluorescence intensity was measured in a circular region of interest in the center of the nucleus, and background-subtracted fluorescence intensities plotted in Python. Trace color inFig. 6 D was assigned according to fluorescence intensity in the last frame of the movie, with respect to the estimated intensity threshold used for flow sorting (dashed line in Fig. 6D).
Cell tracking was carried out with TrackMate (ImageJ) (Tinevez et al., 2017 (link)) based on the constitutively expressed H2B-Cerulean nuclear marker. Fluorescence intensity was measured in a circular region of interest in the center of the nucleus, and background-subtracted fluorescence intensities plotted in Python. Trace color in
5
Tracking Gata6 Expression in Live Cells
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To track Gata6 reporter expression in live cells, PrE-like differentiation was induced by a 6 h pulse of doxycycline-treatment in serum-containing medium as described in (Schröter et al., 2015) (link). 16 hours after doxycycline-removal, cells were either switched directly to N2B27 medium lacking phenol red, or trypsinized, sorted for reporter expression, and seeded on fibronectin-coated imaging dishes (ibidi µ-slides). Time-lapse imaging was started within 2 h after sorting on an Olympus IX81 widefield microscope equipped with LED illumination (pE4000, CoolLED) and a Hamamatsu c9100-13 EMCCD camera. Hardware was controlled by MicroManager software (Edelstein et al., 2001) (link). Time-lapse movies were acquired using a 40x oil immersion lens (NA 1.2), with 10-minute time intervals.
Cell tracking was carried out with TrackMate (Tinevez et al., 2017) (link) based on the constitutively expressed H2B-Cerulean nuclear marker. Fluorescence intensity was measured in a circular region of interest in the center of the nucleus, and background-subtracted fluorescence intensities plotted in Python. Trace color in Figure 4D was assigned according to fluorescence intensity in the last frame of the movie, with respect to the estimated intensity threshold used for flow sorting (dashed line).
Cell tracking was carried out with TrackMate (Tinevez et al., 2017) (link) based on the constitutively expressed H2B-Cerulean nuclear marker. Fluorescence intensity was measured in a circular region of interest in the center of the nucleus, and background-subtracted fluorescence intensities plotted in Python. Trace color in Figure 4D was assigned according to fluorescence intensity in the last frame of the movie, with respect to the estimated intensity threshold used for flow sorting (dashed line).
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