The sorted ILC2 and T-cells were fixed in PBS containing 1% paraformaldehyde, washed with PBS, stored, and protected from light until analysis. Sub-membrane actin and nuclei (DNA) were labeled with Alexa Fluor 488 Phalloidin (Sigma-Aldrich, Munich, Germany) and Hoechst 33258 (Sigma-Aldrich), respectively (32 (link)). The cells were either transferred to optical-bottom 12-well plates (Thermo Fisher Scientific) in PBS for observation by confocal microscopy (32 (link)). The samples were analyzed using a Leica DM6000 inverted microscope connected to a Leica SP5 laser scanning confocal system and Fiji software to determine the cell integrity after sorting.
Sp5 laser scanning confocal system
Manufactured by Leica
The Leica SP5 laser scanning confocal system is a high-performance microscope designed for advanced imaging applications. It features multiple laser excitation sources, a sensitive detection system, and a robust optical design to provide high-quality, high-resolution images of biological samples and materials. The system is capable of performing fast, high-resolution imaging and offers a range of advanced imaging techniques, such as spectral detection and live-cell imaging.
Automatically generated - may contain errors
2 protocols using sp5 laser scanning confocal system
1
Immunocytochemistry of Sorted Cells
2
Confocal Imaging and 3D Modeling of Cellular Colocalization
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Sections of matrix
stones were imaged with a Leica SP5 laser scanning confocal system.
Lower magnification images were acquired with 20× air objective.
Higher magnification images were acquired using a 63.0× (N.A.
1.40) oil objective. For individual channels, imaging was performed
with excitation at 488 nm (51% intensity) and acquisition at 500–600
nm with a gain of 882. The same tissue also was imaged using excitation
at 405 nm (with 27% intensity) and acquisition at 430–550 nm
with a gain of 844.
For image analysis, the Imaris (Bitplane,
v9.7.2) “coloc” module was used to find voxels where
signal was present in both channels (threshold = 2000 for both channels).
A third channel was created using colocalized signals, and surface
objects were created (intensity threshold = 17606). The surface objects
were filtered by size, and all surfaces with less than 2000 voxels
were discarded; the results are shown as a 3D model.
stones were imaged with a Leica SP5 laser scanning confocal system.
Lower magnification images were acquired with 20× air objective.
Higher magnification images were acquired using a 63.0× (N.A.
1.40) oil objective. For individual channels, imaging was performed
with excitation at 488 nm (51% intensity) and acquisition at 500–600
nm with a gain of 882. The same tissue also was imaged using excitation
at 405 nm (with 27% intensity) and acquisition at 430–550 nm
with a gain of 844.
For image analysis, the Imaris (Bitplane,
v9.7.2) “coloc” module was used to find voxels where
signal was present in both channels (threshold = 2000 for both channels).
A third channel was created using colocalized signals, and surface
objects were created (intensity threshold = 17606). The surface objects
were filtered by size, and all surfaces with less than 2000 voxels
were discarded; the results are shown as a 3D model.
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