Wild-type control and Rho knockout eye cups were incubated overnight in TO-PRO-3 stain (Thermo Fisher Scientific; diluted 3000x in PBS). Rho KO eye cups were then washed thoroughly with PBS for 24 hr prior to clearing with a modified iDISCO+ protocol (Renier et al., 2016 (link)). Briefly, eye cups were dehydrated through a methanol/water gradient (20%, 40%, 60%, 80%, 100%, 100%). Incubations were for 30 min at each concentration. Next, eye cups were incubated twice in 100% dichloromethane for 30 min. Finally, eye cups were transferred to 100% ethyl cinnamate and incubated for at least 1 hr prior to imaging. Eye cups were imaged in ethyl cinnamate using a Lightsheet microscope (Zeiss, Jena Germany) with modified optics designed for imaging 1.56 refractive index solutions. A 20 × 1.0 NA objective (RI = 1.56 corrected) was used for detection.
Lightsheet microscope
Manufactured by Zeiss
Sourced in Germany, United Kingdom
The Lightsheet microscope is an advanced imaging system designed for high-resolution, non-invasive observation of living samples. It utilizes a unique illumination technique, known as light-sheet fluorescence microscopy, to capture images with exceptional clarity and contrast. The core function of the Lightsheet microscope is to provide a powerful tool for researchers and scientists to study dynamic biological processes in a wide range of specimens, from single cells to small organisms.
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Lab products found in correlation
5 protocols using lightsheet microscope
1
Rho Knockout Eye Cup Clearing and Imaging
2
Anther Imaging using ClearSeeAlpha
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Anthers fixed in 4% paraformaldehyde were stained in 0.01% (v/v) Renaissance 2200 (Renaissance Chemicals) for 1 h and 10 µg/mL propidium iodide for 1 h. Stained samples were cleared for 48–72 h in ClearSeeAlpha solution44 (link). Samples were embedded in 0.1% agarose gel in a glass capillary. The images were captured using a Lightsheet microscope (Carl Zeiss Z.1) with a 20x Plan Apochromat lens.
3
Spheroid Formation and Light-Sheet Imaging
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1x106 F98 H2B RFP or U-87 MG H2B RFP cells were seeded in 1ml filtered culture media in one well of a 5D spherical plate (Kugelmeiers, Switzerland) and incubated at 37°C overnight for spheroid formation. Spheroids were imaged on a Zeiss Light-Sheet microscope and analyzed using using Imaris v9.6 software (www.imaris.oxinst.com ; Oxford Instruments, UK). See Supplementary Section 1 for full experimental details.
4
Whole Organ Lymph Node Imaging
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Isolated mLN were fixed overnight at 4 °C in freshly prepared 1–2% paraformaldehyde in PBS, washed, and embedded in 2% (w/v) low-melting agarose (Sigma-Aldrich) in PBS. 200 to 500-μm thick sections were cut with a vibratome (Microm HM 650 V) and were used for staining. These thick sections were blocked with blocking buffer (as described above) overnight and stained for at 2 to 5 days with the primary antibodies followed by extensive washing in PBS (total 5×/ 1 h each) before incubation with fluorescently labeled secondary antibodies. After staining, samples were cleared described previously19 (link). After clearing, vibratome sections were imaged using light sheet microscope (Zeiss) with 20×objective in a 80.2% fructose solution. The 3D reconstruction and movies were made using IMARIS (Bitplane). In separate set of experiments, the whole mLN was cleared using X-CLARITY Electrophoretic Tissue Clearing System (Logos) and then processed for sectioning and immunofluorescence staining for 5 to 10 days before imaging on a light sheet microscope. The 3D reconstruction and movies were made using IMARIS.
5
Whole-Mount Imaging of Lymph Nodes
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Isolated mLNs were fixed overnight at 4 C in freshly prepared 1% paraformaldehyde in PBS, washed, and embedded in 2% (w/v) low-melting agarose (Sigma-Aldrich) in PBS. 500-mm sections were cut with a vibratome (Microm HM 650V) and were used for staining. These thick sections were blocked with blocking buffer (as described previously) overnight and stained for at least 96 hr with the primary antibodies listed in Table S2, followed by extensive washing in PBS before incubation with fluorescently labeled secondary antibodies. After staining, samples were cleared using fructose as described previously (Ke et al., 2013) . After clearing, vibratome sections were imaged using light sheet microscope (Zeiss) with 203 objective in a 80.2% fructose solution. The 3D reconstruction and movies were made using IMARIS (Bitplane).
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