To measure nutrient-dependent plasticity of CN axons and presynaptic terminals, adult male flies carrying Crz-Gal4 (or CN-Gal4) and UAS-synaptotagmin (Syt)-GFP were collected 1–2 days after eclosion and recovered for 2–3 more days in standard fly food at 23°C. A group of flies were dissected and their brains were stained by using anti-GFP antibody (fed flies). Another group of flies after 24 hours of starvation (wet starvation) were dissected and their brains were stained similarly (starved flies). We refed some of the starved flies with 200 mM D-glucose (with 1 % agar) for 24 hours and their brains were stained similarly (refed flies). We measured and quantified the number of Syt-GFP+ puncta and the length of axons as described in previous study35 (link). The intensity of Syt-GFP signal was measured and normalized using corrected total fluorescence (CTF) method34 (link), calculated by subtracting the background endogenous fluorescence from the integrated GFP signal of an area of interest. Images were captured by Zeiss LSM 800 confocal microscopy (Zeiss) and were analyzed using ImageJ. Statistical analyses were conducted with GraphPad Prism 8.1.1.
Lsm 800 confocal microscopy
Manufactured by Zeiss
Sourced in Germany, United States
The Zeiss LSM 800 is a confocal microscopy system designed for high-resolution imaging. It features a laser-scanning technology that allows for optical sectioning of samples, enabling the capture of three-dimensional images. The LSM 800 is capable of capturing detailed, diffraction-limited images with improved signal-to-noise ratio and contrast.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Prism 8, by GraphPad (2 mentions)
H2dcfda, by Thermo Fisher Scientific (2 mentions)
Deadend fluorometric tunel system, by Promega (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti insulin antibody, by Cell Signaling Technology (1 mentions)
Slide scanner, by Zeiss (1 mentions)
Guinea pig anti insulin, by Agilent Technologies (1 mentions)
Superfrost slide, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Rhodamine labeled phalloidin, by Yeasen (1 mentions)
H2dcfda, by Zeiss (1 mentions)
Opti mem reduced serum medium, by Thermo Fisher Scientific (1 mentions)
Fluorescence in situ hybridization kit, by RiboBio (1 mentions)
Mitotracker red, by Thermo Fisher Scientific (1 mentions)
Mitochondrial membrane potential assay kit, by Thermo Fisher Scientific (1 mentions)
Dynasore, by Santa Cruz Biotechnology (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Ab97625, by Abcam (1 mentions)
23 protocols using lsm 800 confocal microscopy
1
Nutrient-Dependent Plasticity of Crz Neurons
2
Nutrient-Dependent Plasticity of Crz Neurons
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To measure nutrient-dependent plasticity of CN axons and presynaptic terminals, adult male flies carrying Crz-Gal4 (or CN-Gal4) and UAS-synaptotagmin (Syt)-GFP were collected 1–2 days after eclosion and recovered for 2–3 more days in standard fly food at 23°C. A group of flies were dissected and their brains were stained by using anti-GFP antibody (fed flies). Another group of flies after 24 hours of starvation (wet starvation) were dissected and their brains were stained similarly (starved flies). We refed some of the starved flies with 200 mM D-glucose (with 1 % agar) for 24 hours and their brains were stained similarly (refed flies). We measured and quantified the number of Syt-GFP+ puncta and the length of axons as described in previous study35 (link). The intensity of Syt-GFP signal was measured and normalized using corrected total fluorescence (CTF) method34 (link), calculated by subtracting the background endogenous fluorescence from the integrated GFP signal of an area of interest. Images were captured by Zeiss LSM 800 confocal microscopy (Zeiss) and were analyzed using ImageJ. Statistical analyses were conducted with GraphPad Prism 8.1.1.
3
Pancreatic Tissue Analysis for Insulitis
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Pancreatic tissues were harvested and fixed with 4% PFA overnight at 4 °C and tissues were processed and embedded in paraffin blocks as described previously (80 ). Tissue sections were stained with rabbit anti-insulin antibody (Cell Signaling) and counterstained with DAB peroxidase anti-rabbit IgG (Vector Lab). Images were acquired using a Zeiss slide scanner (Zeiss, Germany), and insulitis scoring was performed on 5 slides, each 30 μm apart, from 9 mice/group as previously described (80 ). Immunofluorescence studies were performed according to the protocol described previously (46 (link)) using the following antibodies: guinea pig anti-insulin (Dako), mouse anti-PD-L1 (Proteintech), and rabbit anti-CXCL10 (Invitrogen). Signals were detected by counter staining with the following antibodies: goat anti-guinea pig (1:400; Invitrogen), goat anti-rabbit (1:200; Invitrogen), and goat anti-mouse (1:200; Invitrogen). All sections were counterstained with DAPI to identify nuclei. Images were obtained with a Zeiss LSM 800 confocal microscopy (Carl Zeiss, Germany), the fluorescence intensities were quantified using Image J, and the corrected fluorescence intensities were presented, as described previously (46 (link)).
4
Immunohistochemical Staining of Nymphal Insect Tissues
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IHC staining was performed as previously described55 (link). Briefly, third-instar nymphs were microinjected with 50 ng dsZfh1. Samples were fixed using 4% paraformaldehyde in PBS overnight at 4 °C, following by blocking with Tissue-Tek O.C.T. Compound (Cat#4583, Sakura Finetek) at −80 °C. Samples were longitudinally cut into ~30 μm sections using a Lecia CM1900 cryotome (Leica Microsystems) at −20 °C, and then transferred to Superfrost+ slides (Cat#12-550-15, Thermo Fisher Scientific). The cytoskeleton and nucleus were stained using 100 nM rhodamine-labeled phalloidin (Cat# 40734ES75, Yeasen) and 100 nM DAPI (Cat#D9542, Sigma Aldrich), respectively. Fluorescence images were acquired using a Zeiss LSM 800 confocal microscopy (Carl Zeiss MicroImaging).
5
ROS Detection using H2DCFDA Assay
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The cellular ROS level was detected using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, Thermo) assay as previously described 41 (link). In brief, cells (5×105 cells/well) were seeded onto glass slides in 6-well plates and incubates at 37 °C overnight. Cells were washed with PBS, followed by incubation with H2DCFDA (50 μM) at 37 °C for 30 min. The cells were washed, paraformaldehyde fixed, permeabilized and stained with DAPI to label nuclei. The fluorescence signals of H2DCFDA were detected by ZEISS LSM800 confocal microscopy (ZEISS, Oberkochen, Germany) and the images were analyzed with Zen2.3 software and ImageJ.
6
Live Cell Imaging of Endosomal Escape
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The Gal-8-based reporter assay to characterize endosomal escape was originally described by Kilchrist et al.,30 (link) and we previously used the B16F10 cancer cell line with the Gal-8-mRuby3 gene integrated using the PiggyBac transposon/transposase system29 (link) to perform endosomal escape assay on fixed cells.8 (link) For live cell imaging, the cells were seeded onto a four-well Nunc Lab-Tek II #1.5 chamber slide (Thermo Fisher Scientific, Cat. #155382) at a density of 0.1 million cells/mL, at 24 h before the experiments. The 50- or 400-nm pDNA/PEIpro NPs carrying 10% Cy5-labeled pDNA were diluted by Opti-MEM reduced serum medium (Gibco, Cat. #31985062) to a concentration of 1 μg pDNA/mL. The original full culture medium, high-glucose DMEM supplemented by 10% fetal bovine serum, was then completely replaced by this NP-containing medium. Then the chamber slide was mounted on a Zeiss LSM800 confocal microscopy equipped with a cell culture chamber. The imaging was carried out under a 63× lens from approximately 30 min after mounting for a total of 4 h. A 101.41 μm × 101.41 μm area was imaged; and a z stack was performed with a slide thickness of 0.27 μm.
7
Visualizing circPTP4A2 Expression
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RNA fluorescence in situ hybridization was performed using a fluorescence in situ hybridization kit (RiboBio, Guangzhou, China) in accordance with the manufacturer's guidelines. Cy3-labeled circPTP4A2 probe (RiboBio, Guangzhou, China) was detected with fluorescence in situ hybridization kit and then observed with LSM800 confocal microscopy (Zeiss, Germany).
8
Monitoring Neuronal Activity in Starved Flies
9
Mitochondrial Morphology Dynamics Assay
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To observe the morphological changes of mitochondria, cells were stained with MitoTracker Red (100 nM; Molecular Probes, ThermoFisher Scientific) at 37°C for 30 min, captured by LSM800 confocal microscopy (Carl Zeiss, Jena, Germany), and analysed using Image J software. Mitochondria were subjected to ‘analyse particles’ to obtain mitochondrial interconnectivity (ratio of area and perimeter) and mitochondrial elongation (ratio of the lengths of major and minor axes), two well‐characterized mediators of mitochondrial fission and fusion as described before.24 At least 100 cells were analysed in each sample to determine cells undergoing mitochondrial fragmentation.
10
Mitochondrial Membrane Potential Assay
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For the detection of mitochondrial membrane potential (ΔΨm), the JC-1 fluorescence with the Mitochondrial Membrane Potential Assay Kit (Invitrogen, Carlsbad, USA) was used in accordance with the manufacturer's instructions. The cells were seeded in 6-well plates at a density of 1 × 105 cells/well, then, incubated with 1 mg/mL JC-1 at 37 °C for 15 min. After washing three times with PBS and covered with 1 μg/mL Hoechst 33342 (Invitrogen, Carlsbad, USA), a Zeiss LSM 800 confocal microscopy was used to detect the fluorescent signals. JC-1 monomers (green) and aggregates (red) fluorescence were observed by excitation at 514 and 585 nm, and emission at 529 and 590 nm, respectively.
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