The frozen sections of tissues were washed by phosphate-buffered saline (PBS), then penetrated with 4% Triton X-100 containing 1% BSA for 20 min. After blocking with goat serum for 1 h at room temperature, tissues were incubated in the primary antibodies at 4 °C overnight followed by incubation with Alexa Fluor 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) or Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 1 h. The sections were observed under a confocal laser scanning microscope (LSM800, Olympus, Tokyo, Japan).
Lsm800
Manufactured by Olympus
Sourced in Japan
The LSM800 is a confocal microscope system designed for high-resolution imaging and analysis of samples. It features an efficient optical design and advanced detection capabilities to deliver clear, detailed images.
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Lab products found in correlation
γh2ax, by Santa Cruz Biotechnology (2 mentions)
β actin, by Proteintech (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
Fv3000, by Olympus (2 mentions)
Alexa fluor 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Propidium iodide staining, by Merck Group (1 mentions)
4 6 diamino 2 fenilindol dapi, by Thermo Fisher Scientific (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Eclipse te2000 confocal microscope, by Nikon (1 mentions)
Fluoview fv10i, by Olympus (1 mentions)
Lysotracker deep red, by Thermo Fisher Scientific (1 mentions)
Fv1200, by Olympus (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Lsm 780, by Olympus (1 mentions)
Axioimager microscope, by Zeiss (1 mentions)
Axiovision software, by Zeiss (1 mentions)
Fv1000, by Olympus (1 mentions)
Anti cxcr4 antibody, by Abcam (1 mentions)
11 protocols using lsm800
1
Tissue Immunofluorescence Staining Protocol
2
Quantifying Cell Viability and Proliferation
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Propidium Iodide (Sigma-Aldrich, Burlington, MA, USA) and Calcein-AM (Invitrogen, Waltham, MA, USA) and were used to assess cell viability after 72 h of culture. Cell containing constructs were washed with 1× PBS (Gibco, Waltham, MA, USA) and stained with Propidium Iodide staining (1 mg/mL; Sigma-Aldrich, Burlington, MA, USA) and Calcein AM (5 μM in PBS 1×; Invitrogen, Waltham, MA, USA) for 30 min at 37 °C and 5% CO2 for 5 min at 37 °C and 5% CO2. Cell proliferation was assessed by immunofluorescence staining incubating the primary antibody Ki67 (1:300, Ref. ab15580, Abcam, Cambridge, UK) overnight. After three rinses in PBS, secondary antibody Alexa Fluor 555 (1:500, Ref. A32723, Invitrogen, Waltham, MA, USA) was incubated for 2 h and cell nuclei were stained with 4′,6-diamino-2-fenilindol (DAPI; 1:1000, Ref.D1306, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). All samples were visualized by fluorescent microscopy (Olympus LSM800, Tokyo, Japan) and images were analyzed by ImageJ program [43 (link)]. Viability and proliferation percentage results were presented as the mean ± standard deviation of 10 random fields in three independent experiments calculated using the following equations:
3
Autophagic flux monitoring in HUVECs
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The HUVECs were transfected with GFP-mRFP-LC3II lentivirus (Hanbio Biotechnology, Shanghai, China) for 1 h and then the medium was replaced with normal medium. After 36 h, the cells were washed with PBS and viewed under a confocal laser scanning microscope (LSM800, Olympus, Tokyo, Japan). GFP and RFP dots were counted by the manual counting of fluorescent puncta in five high-power fields.
4
Immunocytochemical Analysis of DNA Damage
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Immunocytochemistry was performed as described previously51 (link), using antibodies against γH2AX (Santa cruz), 8-OHdG (Nikken Seil), and β-actin (Proteintech). The immunostained samples were observed with either Zeiss LSM800 or Olympus Fluoview FV1000 confocal fluorescence microscope. 8-OHdG intensity was measured in nuclear (DAPI positive areas) and non-nuclear (β-actin positive areas outside of DAPI positive areas) for 20 cells per sample using Image J, and data are shown as mean ± SEM.
5
Immunocytochemistry for γH2AX and β-actin
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Immunocytochemistry was performed as described previously55 (link), using antibodies against γH2AX (Santa cruz) and β-actin (Proteintech), and Fluoro-KEEPER Antifade Reagent (Non-Hardening Type with DAPI, Nakalai) for mounting medium. The immunostained samples were observed with either Zeiss LSM800 or Olympus Fluoview FV1000 confocal fluorescence microscope.
6
Immunostaining Analysis of Cell Lines
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Immunostaining protocols are described in the Supplementary Experimental Procedures . WM samples were analyzed using the Zeiss LSM800 and Olympus FluoView FV10i confocal laser scanning microscopes. iPSCs and inducible-TAp73-Saos-2 samples were analyzed using a Zeiss LSM800 Confocal Laser Scanning Microscope and a Nikon Eclipse TE2000 Confocal Microscope. Confocal z-stack images were taken in all cases. Images were processed using ZEN blue software, FV10-ASW 2.1 viewer software, EZ-C1 software, and ImageJ software.
7
Visualizing S. pneumoniae-Lysosome Interactions
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For the determination of association of S. pneumoniae-containing vesicles with LysoTracker Deep Red by immunofluorescence detection, A549 cells were seeded at 1,5 x 105 cells per well on glass coverslips in 12-well plates. The infection with IAV and S. pneumoniae was performed as described above. Cells were incubated with 1 μM LysoTracker Deep Red (Invitrogen, USA) for 30 min before fixation to stain lysosomes. To analyze the expression of LC3-II in A549 cells infected by IAV, S. pneumoniae, or superinfected, as well as the association between S. pneumoniae and LC3, A549 cells were previously transfected with LC3-mKate2 plasmid using JetPRIME (Polyplus-transfection, Illkirch, France), and 24 h later were infected as described before. In both cases, at 3 hpi with S. pneumoniae, cells were washed 3 times with PBS, fixed with 2% paraformaldehyde– 2% sucrose for 15 min followed by DAPI staining. Finally, coverslips were washed with PBS and mounted on glass slides using Mowiol 4–88. Images were acquired using confocal microscopes (Zeiss LSM-800 and Olympus FV-1200) under 63X oil objectives. Images were processed with ImageJ 1.51n.
8
Transient Transgenesis for Cargo Localization
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For analyses of cargo localization in single neurons, transient transgenesis was utilized. Plasmid DNA encoding the 5kbneurod (pLL neurons; (Mo and Nicolson, 2011 (link)) promotor driving constructs of interest were derived using Gateway technology (Kwan et al., 2007 ). For expression, 3–13 pg of plasmid DNA was microinjected into zebrafish zygotes as previously described (Drerup and Nechiporuk, 2013 (link), 2016 (link)). For analysis, larvae expressing the construct of interest in a subset of pLL neuron cell bodies were selected using a Zeiss AxioZoom fluorescent dissecting microscope. Larvae were anesthetized in 0.02% tricaine and mounted individually in 1.5% low melt agarose in embryo media and imaged with a Zeiss LSM800 or Olympus FV3000 confocal microscope with a 63X/NA1.2 water immersion or 40X/NA1.2 silicone immersion objective respectively. RFP-Rab7, Mito-TagRFP, EB3-GFP, PST1-RFP (peroxisomes), and RFP-Dync1li1v2 constructs for transient transgenesis have been used previously (Drerup et al., 2017 (link); Drerup and Nechiporuk, 2013 (link)). Human LC3 was subcloned for expression from Addgene clone 24920 (Lee et al., 2008 (link)). PatroninCC was subcloned from the Addgene clone 59053 (Hendershott and Vale, 2014 (link)). nudc and lis1b cDNAs were subcloned from zebrafish cDNA and confirmed by sequencing.
9
Quantifying Reactive Oxygen Species in Rice Roots
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The presence of ROS was examined in roots from 10-days old rice seedlings were incubated in 10 μM CM-H2DCFDA for 30 min in dimethyl sulfoxide and in 1 mg/ml of 3, 3′-diaminobenzidine (DAB) solution for 30 min. After being washed three times with PBS, the tissues were observed with Zeiss LSM800 and Olympus BX61 microscopes. Four biological replicates were prepared separately and analyzed three times.
10
Confocal and DIC Imaging Protocols
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Confocal images were acquired using Zeiss LSM 780, LSM 800, and Olympus FV1000 laser scanning confocal microscopes and processed with Zen Blue and Fluoview software. DIC images of in situ hybridizations were collected using a Zeiss Axioimager microscope and Axiovision software.
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