After fixation, devices were permeated with 0.1% Triton-X (Sigma) for 30min and proceeded to incubation with Phalloidin Alexa 488 (1:200, Invitrogen) overnight in the cold room. The devices were washed several times with 1xPBS until fluorescent background was negligible before image acquisition. Confocal images were acquired with 40× water immersion objective, Axiovert 200M inverted microscope (Zeiss), a CSU-X1 spinning disk confocal scan head (Yokogawa Electric Corporation), and iXon3 897 EMCCD camera (Andor Technology). Images were acquired in a tiling mode and stitched using ImageJ (9 ).
Csu x1 spinning disk confocal scan head
Manufactured by Yokogawa
The CSU X1 is a spinning disk confocal scan head designed for high-speed, high-resolution imaging. It utilizes a patented confocal optical system to provide real-time, optically sectioned images.
Automatically generated - may contain errors
Lab products found in correlation
Ix81 inverted fluorescence microscope, by Hamamatsu Photonics (2 mentions)
C9100 13 back thinned em ccd camera, by Yokogawa (2 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Ixon3 897 emccd camera, by Oxford Instruments (1 mentions)
Axiovert 200m inverted microscope, by Zeiss (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
3 protocols using csu x1 spinning disk confocal scan head
1
Actin Cytoskeleton Visualization in Cells
2
Quantitative Fluorescent Microscopy
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Fluorescent images were acquired with an Olympus IX81 inverted fluorescence microscope equipped with a 60× objective (1.35 NA), Hamamatsu C9100-13 back-thinned EM-CCD camera, and Yokogawa CSU X1 spinning disk confocal scan head (with Spectral Aurora Borealis upgrade). Images were acquired of multiple z-slices (0.3 µm) and collapsed xy projections presented. Image analysis was performed using Perkin Elmer Volocity software. Single cells were selected and the Pearson's Correlation calculated for the whole cell using Volocity software.
3
Fluorescent Imaging of Intestinal and Hepatic Cell Markers
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Photomicrographs of immunostained sections of small intestine and liver were taken with an Olympus IX81 inverted fluorescence microscope equipped with a Hamamatsu C9100-13 back-thinned EM-CCD camera and Yokogawa CSU X1 spinning disk confocal scan head. Images were adjusted for contrast and brightness using the Volocity 6.1.1 version software (Perkin Elmer).
Fluorescent confocal images of PNEC/NEB cells immunostained for bombesin, SV2, Mash1, Prox1, HIF1α, or Ki67 in double/triple-immunostained sections were obtained using a Leica confocal laser scanning microscope (model TCS-SPE) and LAS-AF software, as previously reported. 13 The variable excitation wavelengths of the krypton/argon laser were 488 nm for FITC conjugate, 568 nm for Texas Red complex and 695 nm for Alexa Fluor 680 conjugate/RedDot-2 (nuclear counterstaining).
Fluorescent confocal images of PNEC/NEB cells immunostained for bombesin, SV2, Mash1, Prox1, HIF1α, or Ki67 in double/triple-immunostained sections were obtained using a Leica confocal laser scanning microscope (model TCS-SPE) and LAS-AF software, as previously reported. 13 The variable excitation wavelengths of the krypton/argon laser were 488 nm for FITC conjugate, 568 nm for Texas Red complex and 695 nm for Alexa Fluor 680 conjugate/RedDot-2 (nuclear counterstaining).
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