The xenografts (ctrl-sh, MFF-sh or CPT1A-sh2) were embedded in paraffin, sectioned, dehydrated, subjected to antigen retrieval, blocked with 1% BSA for 30 min, and incubated with TOM20 (#13929S, CST) overnight at 4 °C. After washing in 0.1% BSA in PBS, paraffin sections were incubated with secondary antibodies conjugated with Alexa Fluor 594 (Molecular Probes, OR, USA). Cell nuclei were visualized with DAPI. Images were acquired by a confocal laser-scanning microscope (Leica, IL, USA). For the colocalization of DRP1 and mitochondrion, SKOV-3 cells seeded on coverslips were incubated with 50 nM MitoTracker (#9082, CST, USA) for 30 min. After washing in PBS, the cells were fixed in cold methanol and then blocked and incubated with primary antibodies (DRP1, 1:200) overnight. After washes in 0.1% BSA in PBS, cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 (Molecular Probes, OR, USA). Cell nuclei were visualized with DAPI. Images were acquired by a confocal laser-scanning microscope (Leica, IL). For mitochondrial morphology analysis, cells expressing DsRed-mito (Miaoling, Wuhan, China) were infected with lentivirus expressing shRNA or exogenous overexpression plasmids of CPT1A, MFF, MFN2, or Parkin. The infected cells were imaged using a confocal laser-scanning microscope (Leica, IL, USA).
Confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany, United States, Japan, China, United Kingdom
The Leica Confocal Laser Scanning Microscope is an advanced optical imaging system that uses a focused laser beam to scan a specimen and produce high-resolution, three-dimensional images. It combines the principles of fluorescence microscopy and digital image processing to capture detailed information about the internal structure and composition of samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (38 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (32 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (25 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (11 mentions)
Triton x 100, by Merck Group (10 mentions)
Ribo fluorescent in situ hybridization kit, by RiboBio (9 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Fluorescent in situ hybridization kit, by RiboBio (8 mentions)
Microtome, by Leica (7 mentions)
Vectashield, by Vector Laboratories (6 mentions)
Paraformaldehyde (pfa), by Solarbio (6 mentions)
Anti fade reagent, by Solarbio (6 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (5 mentions)
Vectashield mounting medium, by Vector Laboratories (5 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (5 mentions)
Image pro plus, by Media Cybernetics (5 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (5 mentions)
971 protocols using confocal laser scanning microscope
1
Mitochondrial Dynamics in Cancer Xenografts
2
Chlorophyll Fluorescence and ROS Assay
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The chlorophyll fluorescence assay was performed using a Leica confocal laser scanning microscope (laser = 640 nm for chloroplast observation and 488 nm for ROS assay, intensity = 50%, collection bandwidth = 10 nm, gains = 695) according to the manufacturer's instructions. For ROS detection assay, 0.1% (v/v) diaminobenzidine (DAB) A solution was prepared using 50 mM Tris-HCl (pH = 4.5). The leaves of M. micrantha were completely immersed in DAB solution and dyed overnight at 25 °C away from light. Anhydrous ethanol was used for decolorization several times, and pictures were taken when the chlorophyll was completely removed and the leaves were transparent. ROS measurements were performed by the fluorescence assay. Mycelia were stained with 2′,7′-dichloro-dihydro-fluorescein diacetate for 20 min and visualized under a Leica Laser scanning confocal microscope to assess the presence of H2O2.
3
Quantifying Neuronal Synaptic Density in TBI
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Mice were decapitated, then brain was harvested on day 25 after TBI (Figure S2A ). Immunofluorescence staining was conducted as previously described.15 In brief, the brain tissues were fixed in 4% paraformaldehyde at 4°C for 48 h and dehydrated in ethanol and embedded in paraffin. The coronal mouse brain sections were obtained. After dewaxing and rehydration, the sections were treated with 0.01 M citrate buffer (pH 6.0) with 0.1% Tween‐20 at 90–95°C for 5 min for antigen retrieval. The sections were incubated at 4 °C overnight with the mouse anti‐NeuN (1:200; Millipore) and rabbit anti‐PSD95 (1:200; Cell Signaling, Inc.). After washing with PBS, the sections were stained with the secondary antibody, anti‐mouse IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 488 Conjugate) (1:1000; Cell Signaling, Inc.) and anti‐rabbit IgG (H + L), F(ab′)2 Fragment (Alexa Fluor® 594 Conjugate) (1:1000, Cell Signaling, Inc.) for 1 h in the dark at room temperature, and then stained with DAPI (Beyotime Institute of Biotechnology) for 1 min to reveal the nuclei, and analyzed using a laser scanning confocal microscope (Leica). The images were taken using a laser scanning confocal microscope (Leica).
4
Mitophagy and Mitochondrial Morphology Evaluation
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After the various treatments, the DCs were fixed with 4% paraformaldehyde for 20 min at room temperature. To evaluate mitophagy, the fixed DCs were incubated with Mitophagy-Tracker Yellow (Dojindo) for 20–30 min in darkness at room temperature, washed three times with PBS, and then incubated with Lyso-Tracker Green (Dojindo) for 20–30 min in darkness. After being washed with PBS, the DCs were plated onto glass slides, and stained with 4′,6-diamidino-2-phenylindole (DAPI). A laser scanning confocal microscope (Leica, Wetzlar, Germany) was used to observe the cells. To evaluate mitochondrial morphology, fixed DCs were incubated with the dapoxyl probe Mito-Tracker Red (Dojindo) for 20–30 min in darkness at 4 °C. After being washed three times with PBS, the DCs were plated onto glass slides and stained with DAPI. The Leica laser scanning confocal microscope (Leica, Mannheim, Germany) was used to observe the cells (Additional file 3 ).
5
Imaging Exosome Transfer in Co-Culture
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For imaging of RFP-tagged exosome transfer in co-culture, MDA-MB-231/GFP (green fluorescence protein-tranduced MDA-MB-231) cells, RAW264.7/GFP (GFP-transduced RAW264.7) cells, and MDA-MB-231/CD63-RFP cells were mixed (1:1) and cultured for 72 hours. Real-time imaging of RFP-tagged exosome transfer from MDA-MB-231/CD63-RFP cells to MDA-MB-231/GFP cells or RAW264.7/GFP cells was performed by use of a laser scanning confocal microscope (Leica, Wetzlar, Germany). To visualize RFP-tagged exosome uptake by MDA-MB-231/GFP cells or RAW264.7/GFP cells, isolated exosomes were resuspended in phosphate buffered saline (PBS) and quantified by use of the Pierce micro-BCA protein assay kit (ThermoScientific, Waltham, MA, USA). After administration of 10 µg of exosomes into cultured MDA-MB-231/GFP cells or RAW264.7/GFP cells, the cellular uptake of RFP-tagged exosomes was monitored by use of a laser scanning confocal microscope (Leica, Wetzlar, Germany).
6
Cellular Internalization and Subcellular Localization
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Cell internalization tests were conducted as described previously.17 (link) RAW264.7 cells were cultured as described above. Briefly, the cells were inoculated in 6-well plate at a density of 105 cells/well and incubated in a complete medium for 24 h. Then, the cells were washed with D-Hank’s twice, followed by preincubation at 37 °C with one of the following endocytosis inhibitors dissolved in serum-free DMEM: chlorpromazine, cytochalasin D, or fillpin.18 (link) Next, the medium was replaced with complete DMEM containing fluorescent dyes-labeled GAN-OMV (OMV was labeled with DiI and GAN was labeled with FITC) and various endocytosis inhibitors for an additional 4 h. To determine the endocytic pathway through which GAN-OMV is internalized into RAW264.7 cells, the slides were observed under a confocal laser scanning microscope (Leica).
In order to determine the subcellular distribution of OMV, RAW264.7 cells were cultured as described above. Briefly, 105 cells/well were inoculated in 6-well plate in the complete medium for 24 h. Each well of the 6-well plate was pre-placed with glass slides, which facilitate to observe under microscope. After treatment with DiI-labeled OMV for 3.5 h, the cell supernatant was discarded, and the lysosomes were stained with LysoTracker-green (Invitrogen). The slides were taken out and observed under a confocal laser scanning microscope (Leica).
In order to determine the subcellular distribution of OMV, RAW264.7 cells were cultured as described above. Briefly, 105 cells/well were inoculated in 6-well plate in the complete medium for 24 h. Each well of the 6-well plate was pre-placed with glass slides, which facilitate to observe under microscope. After treatment with DiI-labeled OMV for 3.5 h, the cell supernatant was discarded, and the lysosomes were stained with LysoTracker-green (Invitrogen). The slides were taken out and observed under a confocal laser scanning microscope (Leica).
7
Cholesterol Regulation of Mitochondrial Dynamics
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BRIN-BD11 cells were plated in six-well plates containing 25-mm-diameter glass coverslips and cultured overnight. After 12 h of indicated cholesterol treatment with or without the pre-treatment with small molecule M1, complete media was removed, cells were washed with PBS and stained with 200 nM Mito-Tracker Red (M7512) in complete medium at 37 °C for 30 min, washed again with PBS and fixed with 4% paraformaldehyde. The coverslips were then mounted in Vectashield (Vector Laboratories) and imaged in Leica confocal laser scanning microscope (Germany) using Leica advanced fluorescence suite 2.6.3. For GLP-1R internalization study,BRIN-BD11 cells were transfected with GLP-1R-GFP plasmid using Turbofect transfection reagent and plated in six-well plates containing 25-mm-diameter glass coverslips. Sixty hours after transfection, cells in coverslips were incubated with GLP-1-Tmr (GLP-1- Tetra methyl rhodamine) (100 nM) in 200 μl of Krebs-HEPES buffer for 60 min at 4 °C in the dark. The cells were then washed in PBS and incubated at 37 °C for the desired time period in complete medium, after which they were fixed in 4% paraformaldehyde, mounted in Vectashield mounting medium (Vector Laboratories), and imaged using a Leica confocal laser scanning microscope (Germany).
8
Evaluating Cell Behavior on Hydrogel Surfaces
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NIH-3T3 fibroblast (SCSP-515, Stem Cell Bank, Chinese Academy of Sciences, Shanghai, China) cells in a growth phase were treated with trypsin and harvested. According to previous methods54 (link), the cells were seeded on the hydrogels with a density of 5 × 104 cells) in the wells of the tissue culture plates and left undisturbed in an incubator for 3 h to allow for cell attachment. Then, an additional 1 mL of the Dulbecco’s modified eagle medium (HyClone, USA) supplemented with 10% fetal bovine serum (HyClone, USA) was added into each well. The cells were allowed to adhere and grow for 3 and 5 days. The morphologies of the cells on the hydrogel surfaces were observed using a laser scanning confocal microscope (Leica, Germany). The biocompatibility of the hydrogel and the cell proliferation was assessed by the MTT assay. Details were described in Supplementary Notes 25 .
9
Myocardial Oxidative Stress Assessment
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Myocardial ROS steady-state levels were detected by in situ dihydroethidium (DHE) staining 3 hours after reperfusion. In brief, unfixed frozen cross-sections were incubated with DHE staining (Sigma-Aldrich, St Louis, MO, USA) at 37°C for 30 minutes in a humidified chamber protected against light, followed by a wash in phosphate-buffered saline for 5 minutes. The images were confirmed and obtained with a Leica laser scanning confocal microscope. Myocardial superoxide production was measured 3 hours after reperfusion by a lucigenin-enhanced chemiluminescence method as previously described.24 (link) Superoxide production was calculated as relative light units per mg of heart weight per second and presented as the fold change against the sham group.
10
Immunofluorescence Staining of Frozen and Paraffin Sections
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The immunofluorescence method was performed as previously described (76 (link), 77 (link)). Frozen sections (10 μm) were fixed with 4% paraformaldehyde (158127, Sigma Aldrich) for 10 min. Paraffin sections (5 μm) were dewaxed, hydrated and antigen-retrieved by boiling in 10 mM citric acid buffer for 10 min. After permeabilized with 0.1% Triton 100 and blocked with 10% horse serum at 37° C for 1 h, sections were incubated with appropriate dilutions of primary antibodies overnight at 4° C. Primary antibodies used in this study included anti-KI67 (1:200, GB111141, Exilon, Guangzhou), anti-RBPJ (1:200, 14613, Proteintech, Wuhan, China), anti-VANGL2 (1:200, 21492, Proteintech), anti-HAND2 (1:200, sc-9409, Santa Cruz Biotechnogoly, Santa Cruz, USA), anti-IDO1 (1:200, 66528, Proteintech), anti-TPH1 (1:100, CSB-PA024100LA01HU, Cusabio, Wuhan, China). After washing with PBS, sections were incubated with 488-conjugated secondary antibodies (2.5 μg/ml, G21234, Invitrogen, Carlsbad, CA) for 40 min, counterstained with propidium iodide (5 μg/ml, PI, P4170, Sigma-Aldrich) and mounted with Extended™ Diamond Antifade Mountant (Thermo Fisher, Waltham, MA). Images were taken with a laser scanning confocal microscope (Leica, Germany). Each experiment was repeated at least three times.
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