The pancreas was microdissected from Control-Ai14 and Neurod1ST-Ai14 embryos (E18.5) and tissue was processed as described previously178 (link). In addition to tdTomato expression, cleared samples were immunolabeled using anti-insulin and anti-GLP1 antibodies (Supplementary Table 4 ). Zeiss Lightsheet Z.1 microscope with illumination objective Lightsheet Z.1 5x/0.1 and detection objective Dry objective Lightsheet Z.1 5x/0.16 was used for imaging at the Light Microscopy Core Facility of the Institute of Molecular Genetics of the Czech Academy of Sciences. IMARIS software v8.1.1 (Bitplane AG, CA, USA) was used for image processing.
Imaris software v8
Manufactured by Oxford Instruments
Sourced in United States
IMARIS software v8.1.1 is a comprehensive 3D/4D visualization and analysis solution for scientific imaging data. It provides advanced image processing and analysis tools for a wide range of microscopy techniques, including confocal, multiphoton, and light-sheet microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Lightsheet z 1 microscope, by Zeiss (4 mentions)
Lightsheet z 1 5x 0, by Zeiss (2 mentions)
Lsm 780, by Zeiss (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Cm1900, by Leica (1 mentions)
Mounting medium, by Vector Laboratories (1 mentions)
Lightsheet z 1, by Zeiss (1 mentions)
6 protocols using imaris software v8
1
Volumetric Imaging of Pancreas Development
2
Lymph Node Tissue Processing and Immunofluorescence Staining
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Mice were intravascularly perfused with PBS for 2 minutes and, subsequently, with 4% PFA for 8 minutes. Isolated LNs were further fixed in 4% PFA for 3 hours at room temperature. The LNs were equilibrated at 4°C in 10% sucrose solution overnight, followed by 20% sucrose solution for 4 hours and 30% for 2 hours. LNs were cryoembedded, cut into 7 μm–thick sections on a cryostat (CM1900; Leica Biosystems), and mounted onto frosted slides. The slides were blocked with 2% FCS in PBS for 30 minutes using an avidin/biotin blocking kit (Vector Laboratories). The slides were then incubated with primary antibodies (Table 1 ) for 1 hour at room temperature and were further incubated for 30 minutes with appropriate secondary antibodies, counterstained with DAPI, and mounted with mounting medium (Vector Laboratories). Images were obtained with a confocal laser-scanning microscope (LSM780; ZEISS) at room temperature and analyzed with IMARIS software v8.1.1 (Bitplane AG).
3
Quantifying Fungal Burden in Lung Tissue
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IMARIS software v8.1.1 (Bitplane AG, CA, USA) was employed to analyze the images obtained with LSFM. When required, background subtraction was applied in accordance with the diameter of the cell population to eliminate unspecific background signals. Fungal and cellular isosurfaces were segmented using the option “Surface” with a smoothing value of 10% of the expected diameter of the object and manual thresholding of the signal. Filaments were calculated from a masked channel from the A. fumigatus isosurfaces, using starting points of 50 to 70 μm and seed points 7 of 3 μm (depending of the degree of fungal invasion) and manual thresholding of the signal. To measure the distance of the fungal burden from bronchioles, we created isosurfaces from lung epithelial cells (podoplanin) signal and filtered surfaces with a diameter less than 50 μm to exclude alveolus structures.
4
Embryonic Heart Clearing and Imaging
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Embryonic hearts were microdissected from non-diabetic and diabetic control and Hif1aCKO embryos (E16.5 and E18.5). An advanced CUBIC protocol (49 (link)) with some modifications (50 (link)) was used for tissue clearing to enable efficient imaging by light-sheet microscopy. Whole-mount immunohistochemical staining of embryonic hearts was performed, and samples were stored before imaging in Cubic 2 at room temperature. The secondary sympathetic chain was microdissected from non-diabetic and diabetic control-Ai14 and Hif1aCKO-Ai14 embryos (E14.5) and whole-mount stained with NeuN. Zeiss Lightsheet Z.1 microscope with illumination objective Lightsheet Z.1 5×/0.1 and detection objective Dry objective Lightsheet Z.1 5×/0.16 was used for imaging at the Light Microscopy Core Facility of the Institute of Molecular Genetics of the Czech Academy of Sciences. IMARIS software v8.1.1 (Bitplane AG, CA, San Francisco, USA) was used for image processing.
5
Pancreas Clearing and Imaging Protocol
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The pancreas was microdissected from control-Ai14 and Isl1CKO-Ai14 mice (postnatal day P0). We used an advanced CUBIC protocol [65 (link)] for tissue clearing to enable efficient imaging by light-sheet microscopy. Briefly, the microdissected tissue was fixed in 4% PFA for 1 h, washed with PBS, and incubated in a clearing solution Cubic 1 for 5 days at 37 ℃. Before immunolabeling, samples were washed in PBT (0.5% Triton-X in PBS) 4 × for 30 min. In addition to tdTomato expression, cleared samples were immunolabeled using different combinations of antibodies (anti-INS, anti-GLP1, and anti-TUBB3). Samples were stored before imaging in Cubic 2 at room temperature. Zeiss Lightsheet Z.1 microscope with illumination objective Lightsheet Z.1 5x/0.1 and detection objective Dry objective Lightsheet Z.1 5x/0.16 was used for imaging at the Light Microscopy Core Facility of the Institute of Molecular Genetics of the Czech Academy of Sciences. IMARIS software v8.1.1 (Bitplane AG, CA, USA) was used for image processing.
6
Imaging of Sympathetic Chain in Mice
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The secondary sympathetic chain was microdissected from non-diabetic and diabetic, control-Ai14 and Hif1aCKO-Ai14 mice (4 months-of age). We used an advanced CUBIC protocol [57 (link)] for tissue clearing to enable efficient imaging by light-sheet microscopy. Samples were stored before imaging in Cubic 2 at room temperature. Zeiss Lightsheet Z.1 microscope (Carl Zeiss, Germany) with illumination objective Lightsheet Z.1 5x/0.1 and detection objective Dry objective Lightsheet Z.1 5x/0.16 was used for imaging at the Light Microscopy Core Facility of the Institute of Molecular Genetics, CAS, Czechia. IMARIS software v8.1.1 (Bitplane AG, CA, USA) was used for image processing.
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