Images of embryos were captured by Leica MZ FLIII with a GFP LP filter (Leica Microsystems, Wetzlar, Germany). Confocal images were acquired on a Leica TCS-SP8 (Leica). Embryos were mounted in PBS on glass-bottom dishes (IWAKI, Tokyo, Japan). Fluorescence was excited with a 405-nm UV laser for Hoechst 33342, a 638-nm laser for Cy5 or Alexa Fluor 633, a 552-nm laser for Cy3, and a 488-nm laser for Alexa Fluor 488. The hybrid detector HyD (Leica) was used for signal detection. 3D reconstruction was performed by 3D viewer software (Leica) from z-stack images every 5 μm.
3d viewer software
Manufactured by Leica
The 3D viewer software is a tool that allows users to visualize and interact with three-dimensional digital models. It provides the core functionality of loading, rendering, and navigating through 3D data.
Automatically generated - may contain errors
Lab products found in correlation
Tcs sp8, by Leica (2 mentions)
Glass bottom dishes, by Iwaki (2 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (2 mentions)
Mzfliii, by Leica (1 mentions)
Species specific secondary antibodies, by Jackson ImmunoResearch (1 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Tcs sp8 microscope, by Leica (1 mentions)
3 protocols using 3d viewer software
1
Imaging Embryos with Confocal Microscopy
2
Multi-label Confocal Imaging of Embryos and Retinas
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Confocal images were acquired on a Leica TCS-SP8 (Leica). Embryos were mounted in PBS on glass-bottom dishes (Cat # 3971-035; IWAKI, Tokyo, Japan). Fluorescence was excited with a 405 nm UV laser to detect Hoechst 33342, a 552 nm laser to detect Cy3, and a 488 nm laser to detect Alexa Fluor 488. The hybrid detector HyD (Leica) was used for signal amplification. Projection images were created by 3D viewer software (Leica) from z-stack images every 2–4 µm.
Whole retinas were mounted using ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence was excited with a 647 nm laser for Cy5, a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and DAPI. Z-stack images were obtained every 5 µm.
Whole retinas were mounted using ProLong™ Gold Antifade Mountant (Thermo Fisher Scientific). Fluorescence was excited with a 647 nm laser for Cy5, a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and DAPI. Z-stack images were obtained every 5 µm.
3
Whole-Mount Retinal Staining Protocol
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Whole-mounted retinas were stained in accordance with a previous report [21 (link)]. Briefly, enucleated eyes of P7 WT and Exoc3l KO mice
were fixed for 20 min in 4% PFA at RT. A small hole was made in the cornea using a 27 G needle, and a circular incision was introduced using fine scissors. Subsequently, retinal cups were
dissected from the eyes and post-fixed for 30 min in 4% PFA at 4°C. Primary antibodies were rabbit anti-neuron-glial antigen-2 (NG2) (1:200; Millipore-Sigma, St. Louis, MO, USA) and hamster
anti-PECAM1 (1:1000; Abcam, Cambridge, UK ). Secondary antibodies were species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin
coupled to Alexa Fluor dyes (Invitrogen). Nuclei were counterstained with 10 µg/ml Hoechst 33342 (Molecular Probes Inc., Eugene, OR, USA) for 10 min at RT. Whole retinas
were mounted using ProLongTM Gold Antifade Mountant (Thermo Fisher Scientific). Confocal images were acquired under a TCS-SP8 microscope (Leica, Wetzlar, Germany). Fluorescence
was produced using a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI). Z-stack images were obtained
every 5 µm. The hybrid detector HyD (Leica) was used for signal amplification. Projection images were created by 3D viewer software (Leica) from z-stack images.
were fixed for 20 min in 4% PFA at RT. A small hole was made in the cornea using a 27 G needle, and a circular incision was introduced using fine scissors. Subsequently, retinal cups were
dissected from the eyes and post-fixed for 30 min in 4% PFA at 4°C. Primary antibodies were rabbit anti-neuron-glial antigen-2 (NG2) (1:200; Millipore-Sigma, St. Louis, MO, USA) and hamster
anti-PECAM1 (1:1000; Abcam, Cambridge, UK ). Secondary antibodies were species-specific secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or streptavidin
coupled to Alexa Fluor dyes (Invitrogen). Nuclei were counterstained with 10 µg/ml Hoechst 33342 (Molecular Probes Inc., Eugene, OR, USA) for 10 min at RT. Whole retinas
were mounted using ProLongTM Gold Antifade Mountant (Thermo Fisher Scientific). Confocal images were acquired under a TCS-SP8 microscope (Leica, Wetzlar, Germany). Fluorescence
was produced using a 552 nm laser for Cy3, a 488 nm laser for Alexa Fluor 488, and a 405 nm UV laser for Hoechst 33342 and 4’,6-diamidino-2-phenylindole (DAPI). Z-stack images were obtained
every 5 µm. The hybrid detector HyD (Leica) was used for signal amplification. Projection images were created by 3D viewer software (Leica) from z-stack images.
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