Small pieces of shell tissue were cut out close to the suture and fixed and de-coloured according to Pasternak et al. (2015) (link), with minor changes to stain cellulose with calcofluor white. Samples were put into an Eppendorf tube with 1.5 ml of pure MeOH for 20 min at 37 °C. Afterwards the sample was transferred into 0.8 ml of fresh pure MeOH for another 3 min, then 200 µl of dH2O was added in 2 min intervals until reaching 2 ml in total. Following this, samples were washed twice with dH2O for 5 min each. Samples were then transferred on a glass slide, stained with one drop of a ready-to-use calcofluor white stain solution (Sigma-Aldrich) containing 1 g l–1 calcofluor white M2R and 0.5 g l–1 Evans blue, and then mounted on a TCS SP5 II CLSM (Leica Microsystems). As emission source, a 405 nm UV diode was used, and the detection range was set from 450 nm to 500 nm. Pictures were made with the same magnification using a ×40/0.85 objective and a resolution of 0.2 µm.
Clsm tcs sp5 2
Manufactured by Leica
The Leica CLSM TCS SP5 II is a confocal laser scanning microscope (CLSM) designed for advanced imaging applications. It features a modular and flexible architecture, allowing for customization to meet specific research requirements. The CLSM TCS SP5 II provides high-resolution, multi-dimensional imaging capabilities, enabling users to capture detailed information about biological samples.
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Lab products found in correlation
6 protocols using clsm tcs sp5 2
1
Shell Tissue Cellulose Staining Protocol
2
Cellulose Staining in Shell Tissue
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Small pieces of shell tissue were cut out close to suture and fixed and de-coloured according to Pasternak et al. (2015) (link) with minor changes to stain cellulose with calcofluor white. Samples were put into an Eppendorf tube with 1.5 ml pure MeOH for 20 min at 37°C. Afterwards the sample was transferred into 0.8 ml fresh pure MeOH for another 3 min, then 200 µl dH2O was added in 2 min intervals until reaching 2 ml in total. After this, samples were washed twice with dH2O for 5 min each. Afterwards samples were transferred on a glass slide, stained with one drop of a ready-to use calcofluor white stain solution (Sigma-Aldrich) containing 1 g/l calcofluor white M2R and 0.5 g/l evans blue and then mounted on a . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted November 20, 2020. ; https://doi.org/10. 1101/2020.11.20.390906 doi: bioRxiv preprint 16 TCS SP5 II CLSM (Leica Microsystems, Vienna, Austria). As emission source a 405 nm UV diode was used, and detection range was set from 450 to 500 nm. Pictures were made with the same magnification using a 40x/0.85 objective and a resolution of 0.2 µm.
3
Fluorescent Cell Imaging in Brain Slices
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Anesthetized animals were immediately perfused with 2% paraformaldehyde and 2 – 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4). Following removal, brains were sliced at 80 μm parallel to the area flattened by the cranial window. Slices were quickly imaged at low magnification (20x, 0.848 × 0.848 μm/pixel) using a Leica CLSM TCS SP5 II running LAS AF (ver. 3.0, Leica) with 488 nm laser excitation. Fluorescence of GCaMP6s was used to locate the target cell in this view. Autofluorescence resulting from glutaraldehyde fixation also produced signal within the tissue slices. A field of view large enough to cover roughly one-fourth of the slice, with the target cell included, was captured using image tiling. This process was performed to identify the fluorescent cell of interest and for slice-level correlation in later steps of the workflow. The slice containing the target cell was then imaged with the same 20x objective at higher pixel resolution (0.360 × 0.360 × 0.976 μm/voxel) to obtain a z-stack of the full depth of the slice and immediate region surrounding the target cell. CLSM imaging required approximately 1-3 hours to find the cell of interest within a slice and capture a tiled slice overview and higher resolution z-stack.
4
Fluorescent Cell Imaging in Fixed Brain Slices
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Anesthetized animals were immediately perfused with 2% paraformaldehyde and 2 – 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4). Following removal, brains were sliced at 80 µm parallel to the area flattened by the cranial window. Slices were quickly imaged at low magnification (20x, 0.848 × 0.848 µm/pixel) using a Leica CLSM TCS SP5 II running LAS AF (ver. 3.0, Leica) with 488 nm laser excitation. Fluorescence of GCaMP6s was used to locate the target cell in this view. Autofluorescence resulting from glutaraldehyde fixation also produced signal within the tissue slices. A field of view large enough to cover roughly one-fourth of the slice, with the target cell included, was captured using image tiling. This process was performed to identify the fluorescent cell of interest and for slice-level correlation in later steps of the workflow. The slice containing the target cell was then imaged with the same 20x objective at higher pixel resolution (0.360 × 0.360 × 0.976 µm/voxel) to obtain a z-stack of the full depth of the slice and immediate region surrounding the target cell. CLSM imaging required approximately 1–3 hours to find the cell of interest within a slice and capture a tiled slice overview and higher resolution z-stack.
5
Imaging Fluorescent Neurons in Fixed Brain Slices
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Anesthetized animals were immediately perfused with 2% paraformaldehyde and 2 – 2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4). Following removal, brains were sliced at 80 μm parallel to the area flattened by the cranial window. Slices were quickly imaged at low magnification (20x, 0.848 × 0.848 μm/pixel) using a Leica CLSM TCS SP5 II running LAS AF (ver. 3.0, Leica) with 488 nm laser excitation. Fluorescence of GCaMP6s was used to locate the target cell in this view. Autofluorescence resulting from glutaraldehyde fixation also produced signal within the tissue slices. A field of view large enough to cover roughly one-fourth of the slice, with the target cell included, was captured using image tiling. This process was performed to identify the fluorescent cell of interest and for slice-level correlation in later steps of the workflow. The slice containing the target cell was then imaged with the same 20x objective at higher pixel resolution (0.360 × 0.360 × 0.976 μm/voxel) to obtain a zstack of the full depth of the slice and immediate region surrounding the target cell. CLSM imaging required approximately 1–3 hours to find the cell of interest within a slice and capture a tiled slice overview and higher resolution z-stack.
6
High-Resolution Imaging of Fluorescent Cells
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Anesthetized animals were immediately perfused with 2% paraformaldehyde and 2-2.5% glutaraldehyde in a 0.1 M sodium cacodylate buffer (pH 7.4). Following removal, brains were sliced at 80 μm parallel to the area flattened by the cranial window. Slices were quickly imaged at low magnification (20×, 0.848 × 0.848 μm/pixel) using a Leica CLSM TCS SP5 II running LAS AF (ver. 3.0, Leica) with 488 nm laser excitation. Fluorescence of GCaMP6s was used to locate the target cell in this view. Autofluorescence resulting from glutaraldehyde fixation also produced signal within the tissue slices. A field of view large enough to cover roughly one-fourth of the slice, with the target cell included, was captured using image tiling. This process was performed to identify the fluorescent cell of interest and for slicelevel correlation in later steps of the workflow. The slice containing the target cell was then imaged with the same 20× objective at higher pixel resolution (0.360 × 0.360 × 0.976 μm/voxel) to obtain a z-stack of the full depth of the slice and immediate region surrounding the target cell. CLSM imaging required approximately 1-3 h to find the cell of interest within a slice and capture a tiled slice overview and higher resolution z-stack.
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