A Leica SP5, SP8 X (Leica Biosystems Inc., Buffalo Grove, IL), or Ultramicroscope II (LaVision BioTec, Bielefeld, Germany) were used to image all relevant structures. To allow for confocal imaging of DBE-cleared tissues, a chamber was manufactured in-house. 3-D overview images were generated of the spine and spinal cord in order to reveal PRV labeling in preganglionic sympathetic neurons (in the IML and medially) and DRG. The spine was repositioned with the ventral side up to allow for optimal imaging of thoracic and lumbar sympathetic chain ganglia, as well was the celiac ganglion. Lightsheet imaging was performed with a zoom factor of 0.63x or 1.6x (specified in figure captions). Images of TH and PRV labeling were collected for all samples, while CD31 staining was not performed for all samples. Images from the far-red fluorescent channel were pseudocolored as green. Additionally, higher magnification (10x/0.40 NA) confocal images captured of PRV-positive sympathetic chain ganglia, their corresponding DRG, and PRV-positive preganglionic cell bodies. A detailed experimental protocol is available through Protocols.io (https://www.protocols.io/edit/light-sheet-microscopy-wz3ff8n ).
Sp8 x
Manufactured by Leica
Sourced in Germany
The Leica SP8 X is a confocal laser scanning microscope designed for advanced imaging applications. It features a range of laser options and high-performance detectors to capture detailed images of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ultramicroscope 2, by Miltenyi Biotec (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Las x software, by Leica (1 mentions)
A11031, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Proteintech (1 mentions)
β catenin d10a8 xp, by Cell Signaling Technology (1 mentions)
Tom20 11802 1 ap, by Proteintech (1 mentions)
Tc coverslip 13 mm st cs200, by Thermo Fisher Scientific (1 mentions)
A11008, by Thermo Fisher Scientific (1 mentions)
E cadherin, by ABclonal (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fluoro keeper antifade reagent, by Nacalai Tesque (1 mentions)
5 protocols using sp8 x
1
Light Sheet Microscopy of Sympathetic Nervous System
2
Light Sheet Microscopy of Cleared Tissues
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A Leica SP5, SP8 X (Leica Biosystems Inc., Buffalo Grove, IL), or Ultramicroscope II (LaVision BioTec, Bielefeld, Germany) were used to image all relevant structures. To allow for confocal imaging of DBE‐cleared tissues, a chamber was manufactured in‐house. Three‐dimensional (3D) overview images were generated of the spine and SC in order to reveal PRV labeling in preganglionic sympathetic neurons (in the IML and medially) and DRG. The spine was repositioned with the ventral side up to allow for optimal imaging of thoracic and lumbar SChG, as well was the CG. Lightsheet imaging was performed with a zoom factor of 0.63× or 1.6× (specified in figure captions). Images of TH and PRV labeling were collected for all samples, while CD31 staining was not performed for all samples. Images from the far‐red fluorescent channel were pseudocolored as green. Additionally, higher magnification (10×/0.40 NA) confocal images captured of PRV‐positive SChG, their corresponding DRG, and PRV‐positive preganglionic cell bodies. A detailed experimental protocol is available through Protocols.io (https://www.protocols.io/edit/light-sheet-microscopy-wz3ff8n ).
3
Visualizing Biofilm Growth on Coupons
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Biofilm growth on coupons was visualized using confocal microscopy. Visualization of biofilms was performed with SYBR Green staining. Coupons were washed in PBS and transferred to a 12-well glass-bottom plate containing 500 µL of PBS and incubated in the presence of 2 µL of 100× concentrated SYBR Green for 15 min in the dark at RT. Specimens were imaged using a confocal laser scanning microscope (Leica SP8X) with a 10× lens (Leica Biosystems, Nussloch, Germany). During observation, the excitation wavelength was 488 nm, and the emission wavelength range used for detecting SYBR Green was 501–548 nm. Photographs were obtained and then analyzed with Leica LAS X software.
4
Immunofluorescence Staining of CRC Cells
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CRC cells seeded on coverslips (Sarstedt Inc. TC coverslip 13 mm ST/CS200, Fisher Scientific) were fixed with 4% PFA in PBS for 20 min at rt. After washing three times with 0.2% BSA in Dulbecco's phosphate-buffered saline (DPBS), the cells were permeabilized with 0.1% Triton X-100 in PBS for 7 min. The permeabilized cells were blocked with DPBS containing 0.2% bovine serum albumin (BSA) for 30 min and then incubated with primary antibody (1:100-2000) overnight at 4°C, followed by Alexa-conjugated secondary antibody (1:200) incubation for 1 h at rt. The coverslips were mounted in DPBS supplemented with DABCO. All IF images were acquired with a laser scanning confocal microscope (Leica SP8 X, Leica) equipped with LAS X software, using a 63x3 1.3 NA oil objective. The following antibodies were used: mSHMT (sc-390641, Santa Cruz Biotechnology), TOM20 (11802-1-AP, Proteintech), β-catenin (D10A8) XP (8480, Cell Signaling Technology), E-cadherin (A11509, ABclonal), vimentin (10366-1-AP, Proteintech), goat anti-rabbit IgG (H+L) cross-adsorbed Alexa Fluor 488 (A-11008, Invitrogen), and goat anti-mouse IgG (H+L) cross-adsorbed Alexa Fluor 568 (A-11031, Invitrogen).
5
Whole-mount Immunostaining of Gastruloids
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For whole-mount immunostaining, gastruloids were fixed in 4% paraformaldehyde overnight at 4 °C, washed with PBST (0.2% Tween20), soaked in blocking buffer (PBS containing 0.1% BSA and 0.3% Triton X-100) overnight at 4 °C and then incubated with primary antibodies diluted with blocking buffer overnight at 4 °C. The samples were washed with washing buffer (PBS containing 0.3% Triton X-100), incubated with secondary antibodies and DAPI overnight at 4 °C, and washed and mounted in Fluoro-KEEPER antifade reagent (Nacalai USA). For phalloidin staining, Alexa Fluor™ 647 Phalloidin (A22287; Thermo) was added to the secondary antibody at a dilution of 1:400. All samples were analyzed with an LSM710 (Zeiss) or LEICA SP8X (Leica) confocal microscope with negative control samples where the primary antibodies were not added. The antibodies used in this study are listed in Table S8 .
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