Tissues were harvested after perfusing Il21-mCherry/Il2-emGFP TG mice with 4% paraformaldehyde (PFA, pH 7.4 at 4 °C), tissues were further fixed in 4% PFA at 4°C for 2 hours, placed in 30% sucrose for 24 hours, and embedded in OCT compound (Tissue-Tek) for freezing. 10-µm thick frozen tissue sections were rehydrated, blocked with 10% rat serum in 0.1% Tween20/PBS (PBST) for 1 hour at room temperature, and stained with the Alexa Fluor 647 anti-mouse B220 antibody (clone RA3–6B2, Biolegend) in 1% rat serum/ 0.1% PBST overnight at 4 °C. Nuclei were stained with 4’,6-diamidin-2-fenilindolo (DAPI). Images were acquired using a Leica TCS SP5 microscope (Leica Microsystems, Mannheim, Germany) using a 20x objective NA .7, zoom 1X and processed with Imaris software 9.0.0 (Bitplane AG, Zurich Switzerland).
Imaris software 9
Manufactured by Oxford Instruments
Sourced in United Kingdom, Switzerland
Imaris software 9.0.0 is a comprehensive imaging and analysis software for 3D and 4D data. It provides advanced tools for visualization, segmentation, and quantification of complex biological samples.
Automatically generated - may contain errors
6 protocols using imaris software 9
1
Visualization of IL-21 and IL-2 Expression
2
Visualization of IL-21 and IL-2 Expression
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Tissues were harvested after perfusing Il21-mCherry/Il2-emGFP TG mice with 4% paraformaldehyde (PFA, pH 7.4 at 4 °C), tissues were further fixed in 4% PFA at 4°C for 2 hours, placed in 30% sucrose for 24 hours, and embedded in OCT compound (Tissue-Tek) for freezing. 10-µm thick frozen tissue sections were rehydrated, blocked with 10% rat serum in 0.1% Tween20/PBS (PBST) for 1 hour at room temperature, and stained with the Alexa Fluor 647 anti-mouse B220 antibody (clone RA3–6B2, Biolegend) in 1% rat serum/ 0.1% PBST overnight at 4 °C. Nuclei were stained with 4’,6-diamidin-2-fenilindolo (DAPI). Images were acquired using a Leica TCS SP5 microscope (Leica Microsystems, Mannheim, Germany) using a 20x objective NA .7, zoom 1X and processed with Imaris software 9.0.0 (Bitplane AG, Zurich Switzerland).
3
Quantitative Analysis of Synaptic Markers in Drosophila
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All images of adult brains were obtained using a Zeiss LSM880 Laser Scanning Confocal Microscope (Carl Zeiss, Oberlochen, Germany) using a 20X 0.8 NA Plan-Apochromat lens, 40X 1.4 NA Plan-Apochromat lens, or a 63X 1.4 NA Plan-Apochromat f/ELYRA lens at an optical zoom of 3x. Images of third instar larval NMJs were obtained using the same confocal microscope using a 40X 1.4 NA Plan-Apochromat lens or a 63X 1.4 NA Plan-Apochromat f/ELYRA lens. Images were centered on the glomerulus or NMJs of interest and the z-boundaries were set based on the appearance of the synaptic labels, Brp-Short-mStraw or mCD8-GFP. Images were analyzed three dimensionally using the Imaris Software 9.7.1 (Oxford Instruments, Abingdon, UK) on a custom-built image processing computer (Digital Storm, Fremont, CA) following previously established methods (Aimino et al., 2023 (link)). For both adult brains and larval NMJs, Brp-Short and endogenous Brp puncta were quantified using the “Spots” function with a spot size of 0.6 μm. Neurite volume was quantified using the “Surfaces” function with a local contrast of 3 μm and smoothing of 0.2 μm for Or47b ORNs. The resultant masks were then visually inspected to ensure their conformation to immunostaining.
4
Quantitative 3D Analysis of Adult Brains and Larval NMJs
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All images of adult brains were obtained using a Zeiss LSM880 Laser Scanning Confocal Microscope (Carl Zeiss, Oberlochen, Germany) using a 20× 0.8 NA Plan-Apochromat lens, 40× 1.4 NA Plan-Apochromat lens, or a 63× 1.4 NA Plan-Apochromat f/ELYRA lens at an optical zoom of 3×. Images of third instar larval NMJs were obtained using the same confocal microscope using a 40× 1.4 NA Plan-Apochromat lens or a 63× 1.4 NA Plan-Apochromat f/ELYRA lens. Images were centered on the glomerulus or NMJs of interest, and the z-boundaries were set based on the appearance of the synaptic labels, Brp-Short-mStraw or mCD8-GFP. Images were analyzed 3 dimensionally using the Imaris Software 9.7.1 (Oxford Instruments, Abingdon, UK) on a custom-built image processing computer (Digital Storm, Fremont, CA, USA) following previously established methods (Aimino et al. 2023 (link)). For both adult brains and larval NMJs, Brp-Short-mStraw and endogenous Brp puncta were quantified using the “Spots” function with a spot size of 0.6 µm. Neurite volume was quantified using the “Surfaces” function with a local contrast of 3 µm and smoothing of 0.2 µm for Or47b olfactory receptor neurons (ORNs). The resultant masks were then visually inspected to ensure their conformation to immunostaining.
5
Quantifying Synaptic Puncta and Neurite Volume
6
Quantifying Synaptic and Neuronal Architecture
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All images were obtained using a Zeiss LSM880 Laser Scanning Confocal Microscope (Carl Zeiss, Oberlochen, Germany) using a 40X 1.4 NA Plan-Apochromat lens or a 63X 1.4 NA Plan-Apochromat f/ELYRA lens at an optical zoom of 3x. Images were centered on the glomerulus of interest and the z-boundaries were set based on the appearance of the synaptic labels, Brp-Short-mStraw and mCD8-GFP. Images were analyzed three dimensionally using the Imaris Software 9.3.1 (Oxford Instruments, Abingdon, UK) on a custom-built image processing computer (Digital Storm, Fremont, CA) following previously established methods (Mosca and Luo, 2014; Mosca et al., 2017) . Brp-Short puncta were quantified using the "Spots" function with a spot size of 0.6 µm. Neurite volume was quantified using the "Surfaces" function with a local contrast of 3 µm and smoothing of 0.2 µm for AM29, Or47b, and Or67d ORNs or a local contrast of 0.5 and a smoothing of 0.2 µm for Mz19 PNs and NP3056 LNs. The resultant masks were then visually inspected to ensure their conformation to immunostaining. Images were processed using ImageJ (NIH, Bethesda, MD) and Adobe Photoshop 2020 (Adobe Systems, San Jose, CA). Figures were produced using Adobe Illustrator CC 2019 (Adobe Systems, San Jose, CA).
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