Whole-mount samples were stained and cleared with a modified 3DISCO protocol37 (link). In short, samples stored in PBS-GT were incubated with primary antibodies in PBS-GT with shaking, for 36 h at RT. Excessive antibody was removed by thorough washing in PBS-GT for 6–12 h and refreshing the solution every 1–2 h. Incubation with fluorophore-coupled secondary antibodies (Molecular Probes) in PBS-GT for 36 h was followed by thorough washing in PBS-GT as described above. When necessary, samples were dehydrated in an ascending Tetrahydrofuran (Sigma, #186562) series (50%, 70%, 3 × 100%; 60 min each), and subsequently cleared in dichloromethane (Sigma, #270997) for 30 min and eventually immersed in benzyl-ether (Sigma, #108014). Non-cleared samples were imaged in 35 mm glass-bottom dishes (Ibidi, #81218) using a laser scanning confocal microscope (Zeiss LSM710) or SP8 Multiphoton microscope (Leica). Cleared samples were imaged whilst submerged in benzyl-ether with a light-sheet fluorescence microscope (LaVision BioTec).
Light sheet fluorescence microscope
Manufactured by Miltenyi Biotec
Sourced in Germany
The Light-sheet fluorescence microscope is a high-resolution imaging tool that employs a planar illumination technique to capture images of biological samples. It illuminates the sample with a thin sheet of light, allowing for optical sectioning and minimizing photodamage to the specimen. The light-sheet approach enables rapid, high-contrast imaging of large samples, making it a valuable tool for various applications in the life sciences.
Automatically generated - may contain errors
3 protocols using light sheet fluorescence microscope
1
3DISCO Whole-Mount Sample Staining
2
Quantitative Lung Vascular Permeability Imaging
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3 h after AAPV induction, mice were injected i.v. with 50 µg Texas red–labeled albumin in 100 μl PBS. 1 h later, animals were killed in CO2/air (50/50 [vol/vol]) and exsanguinated by an incision in the right femoral artery. Mice were perfused in PBS through the left ventricle at a flow speed of 10 ml/min for 90 s. Lungs were subsequently explanted and fixed in 4% methanol-free PFA overnight at 4°C.
Optical clearing was achieved as previously described (Klingberg et al., 2017 (link)) by transfer of the samples into 99% ethanol for 3 h at room temperature. Samples were then transferred into ethyl cinnamate and incubated for a further 5 h. Image stacks of the cleared lungs were acquired with a step size of 20 µm using a custom build light-sheet-fluorescence-microscope (LaVision Biotec). Samples were excited with two light sheets at 561 nm and recorded using an emission filter at 620/60 nm to detect albumin depositions. Texas red–labeled albumin deposition was quantified in FIJI-Image J by physician-aided thresholding, resulting in the scale bar within the picture. Thresholds were set to equal values for all samples. Files were exported in nrrd format and imported into ParaView v5.7.0 for 3D reconstruction.
Optical clearing was achieved as previously described (Klingberg et al., 2017 (link)) by transfer of the samples into 99% ethanol for 3 h at room temperature. Samples were then transferred into ethyl cinnamate and incubated for a further 5 h. Image stacks of the cleared lungs were acquired with a step size of 20 µm using a custom build light-sheet-fluorescence-microscope (LaVision Biotec). Samples were excited with two light sheets at 561 nm and recorded using an emission filter at 620/60 nm to detect albumin depositions. Texas red–labeled albumin deposition was quantified in FIJI-Image J by physician-aided thresholding, resulting in the scale bar within the picture. Thresholds were set to equal values for all samples. Files were exported in nrrd format and imported into ParaView v5.7.0 for 3D reconstruction.
3
Light-sheet Fluorescence Microscopy of Arteries
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Arteries were observed under the light-sheet fluorescence microscope (LaVision Biotec, Bielefeld, Germany). We used a 2.5x objective lens (Mv PLAPO 2VC, Olympus) or a 4x objective lens (Mv PLAPO 2VC, Olympus) covered with a 6mm working distance dipping cap. A supercontinuum white light laser (SuperK EXTREME 80 mHz VIS with wavelength from 400 to 2400nm, NKT Photonics, Cologne, Germany) was chosen as a laser source. To observe the cell distribution and artery morphology, the filters were set as 551/40nm excitation and 567/50nm emission for PKH26 and 640/30nm excitation and 690/50nm emission for Evans blue. The step size was set to 5µm. Carotid artery scanning range was set to 1mm. 3D projections of the tagged image file format (TIFF) images of the artery were obtained by using Imaris software (Bitplane, Oxford Instruments Company).
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