Cells were fixed with 4% PFA at RT for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 2 min. For blocking, cells were incubated with 2% FBS in PBS for 1 hr at RT. Cells were then incubated at 4°C overnight with the following primary antibodies: Tbc1d24 (ProteinTech, 25254-1-AP, 1:200, LSBio, LS-C679739, 1:200, Washington, US), Vimentin (Santa Cruz, sc-6260, 1:100, Texas, US), Impdh (Santa Cruz, sc-36-5171, 1:100, ProteinTech, 12948-1-AP, 1:100), Cpts1 (ProteinTech, 15914-1-AP, 1:200 and LSBio, LS-C197001, 1:100). After washing with PBS, cells were incubated with anti-mouse IgG conjugated with Alexa Fluor 546 (Invitrogen, A11030, 1:1000) or anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen, A11008, 1:1000) for 1 hr at RT. Prolong Gold Antifade mountant with DAPI (Invitrogen) was used for mounting and nuclear staining. Cellular images were taken with a confocal microscope (Leica, TCP SP8, Wetzlar, Germany) or a super-resolution fluorescence microscope (GE healthcare, DeltaVision Elite). The length (μm) of cytoophidia was measured using Fiji [39 (link)].
Tcp sp8
Manufactured by Leica
Sourced in Germany
The TCP SP8 is a high-performance confocal laser scanning microscope from Leica. It is designed for advanced imaging applications, offering exceptional image quality and versatility. The system features a modular architecture, allowing for customization to meet the specific needs of researchers and microscopy users.
Automatically generated - may contain errors
Lab products found in correlation
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Tbc1d24, by Proteintech (1 mentions)
Deltavision elite, by GE Healthcare (1 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ngf 2.5s, by Thermo Fisher Scientific (1 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Anti β tubulin antibody, by Cell Signaling Technology (1 mentions)
10 protocols using tcp sp8
1
Immunofluorescence Staining of Cellular Structures
2
FRET Analysis of Protein Interactions
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FRET studies were carried out as described by Zhang et al. (2017).19 (link) GFP and mCherry fluorescence were observed by a Leica TCP SP8 FRET-FLIM confocal microscope (Leica TCP SP8) with 65 mW argon lasers and a 20×/0.75 NA objective lens. For the FRET signal, GFP fluorescence was excited by the 8% 488 nm laser and collected over 500 to 535 nm with 30% gain value. Meanwhile, mCherry fluorescence was collected over 590 to 645 nm with 100% gain value. mCherry also was separately excited by the 8% 552 nm laser as a mCherry control. The FRET efficiency was calculated as described in the figure legends. For the photobleaching experiments, five scans were taken with the 8% 488 nm laser to provide the pre-bleach data, before photobleaching for 50 seconds with 100% 552 nm light followed by five scans with the 8% 488 nm laser to obtain the post-bleach data. The FRET efficiency was calculated as described in the figure legends.
3
Localization of MeSSIII-1 Fusion Protein
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The CDS of MeSSIII-1 without stop codons was amplified by PCR with the 1300-MeSSIII-1-F/R primers containing Kpn I and Sal I restriction sites (Table S1 ). The amplified product was recombined into pCAMBIA1300-35S-GFP by homologous recombination method to form the recombinant plasmid pCAMBIA1300-35S-MeSSIII-1::GFP. The fusion vector pCAMBIA1300-MeSSIII-1-GFP and empty vector pCAMBIA1300-35S-GFP(control) was transformed into epidermal cells from tobacco (Nicotiana benthamiana) in the 4–6 leaves stage, using the Agrobacterium tumefaciens strain LBA4404 [49 (link)]. They were incubated for 48 h in a greenhouse at 22 °C under a 16 h light/8 h dark cycle at 50% relative humidity. The GFP fluorescence (excitation 488 nm, emission 500–550 nm) and chlorophyll autofluorescence (excitation 640.6 nm, emission 663–738 nm) were observed under a confocal microscope (LEICA, TCP SP8, Wetzlar, Germany).
4
Bimolecular Fluorescence Complementation Assay
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The ORFs of PsF-box1, PsF-box1N, and PsF-box1C were inserted into 35S-SYPCE (CE) vector, and the PsRGL1 ORF was ligated into 35S-SPYNE173 (NE) vector according to the method described by Ye et al. [57 (link)]. Nicotiana benthamiana leaf after infection for 48 h was used to observe the fluorescence distribution using a laser confocal microscope (TCP SP8; Leica, Germany). The primers used for BiFC are listed in Table S1 (see online supplementary material).
5
Subcellular Localization of PsRGL1 and PsF-box Proteins
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The open reading frames (ORFs) of PsRGL1, PsF-box1, PsF-box2, and PsF-box3 without a stop codon were amplified to construct PsRGL1-GFP, PsF-box1-GFP, PsF-box2-GFP, and PsF-box3-GFP fusion constructs. These constructs were then transformed into Agrobacterium GV3101 and infected to Nicotiana benthamiana leaves. DAPI was used to stain the nucleus, and the fluorescence distribution was observed using a laser confocal microscope (TCP SP8; Leica, Germany). The primers used for the subcellular localization are listed in Table S1 (see online supplementary material).
6
Labeling and Culturing Dorsal Root Ganglia
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Chick embryos at E10 were sacrificed by decapitation, visceral organs and tissues were removed, and lumbar DRGs were collected in ice-cold PBS [36] (link). After clearance of peripheral tissue, DRG were labeled with Molday ION-Evergreen reagent (2 mg/ml, BioPAL, Worcester, MA, USA) for 24 hr. After PBS rinses, they were cut into halves, and each half was placed on the surface of SDS-decellularized human lenticules (protocol in earlier section) and incubated in DMEM/F12 medium (Invitrogen) supplemented with 2% fetal bovine serum (FBS, Invitrogen) and nerve growth factor (NGF 2.5S, at 5 and 50 ng/ml, respectively; Thermo Fisher, Waltham, MA, USA) for 5 days. The samples were fixed and stained for TuJ1 and phalloidin, followed by fluorescence-conjugated secondary antibodies, and examined under laser-scanning confocal microscopy (TCP SP8, Leica, Wetzlar, Germany). Serial z-stack images (1 μm thickness) were collected and 3D-reconstructed using LAS X LSC software (Leica).
7
Cellular Uptake and Distribution of DOX-Loaded Micelles
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The cell uptake and drug distribution of the DOX-loaded micelles were determined by confocal laser scanning microscopy (CLSM). For CLSM studies, HeLa cells were incubated on 35 mm diameter glass dishes at a cell density of 1 × 104 mL−1. Subsequently, cells were incubated with DOX·HCl and DOX-loaded micelles at a final concentration of 10 μg mL−1 for 4 h at 37 °C. The culture medium was removed and the cells were rinsed three times with PBS. Subsequently, the images were captured by using a Leica TCP SP8 (Leica, Wetzlar, German).
8
Microtubule Visualization in HT-29 and HeLa Cells
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HT-29 and HeLa cells were cultured on coverslips and were treated with 0.1% DMSO alone and either with 30 nM OAT-449 or 30 nM vincristine in 0.1% DMSO, in triplicate. After 24 h of treatment, the cells were fixed with a PEM buffer (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, 3% sucrose, 0.1% glutaraldehyde, 4% formaldehyde, pH 6.8) for 15 min and permeabilized with 0.5% Triton-X-100 for another 15 min at room temperature. The staining was performed using an anti-β-tubulin antibody from Cell Signaling Technology (Danvers, MA, USA) according to the manufacturer’s protocol. The cells were incubated with the anti-β-tubulin antibody overnight at 4 °C and, after washing, with a secondary antibody conjugated to Alexa Fluor 488. The staining results were observed under a confocal microscope Leica TCP SP8. Obtained images were analyzed using Leica Application Suite X 1.1.0.12420 software.
9
Transient Expression of Fluorescent Markers
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The pCambia1300-NtCIPK23-GFP plasmid, PM (Plasma membrane) marker pm-rk CD3-1007 and pGDp19 were transformed into A. tumefaciens EHA105, and were then infiltrated into leaves of N. benthamiana, as described previously [29 (link)]. Pictures were captured with confocal microscope (Leica TCP SP8, Leica Microsystems, Germany), 48 h after infiltration. The GFP was excited at 488 nm and its emission was captured at 550–590 nm [47 (link)]. The mCherry was detected at 543 nm and its emission was captured at 570–600 nm.
10
Oxidative Stress and Aortic Lesions
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ROS level in the cryostat aortic cross sections was measured with a confocal microscopy using dihydroethidium (DHE) dye as described [ ], and incubated with DHE (5 μmol/L, Invitrogen, Carlsbad, CA, USA) at 37 °C for 15 min in normal saline. After washing with normal saline, the preparations were examined using a laser scanning confocal microscope (Leica TCP SP8, Leica Microsystems). The fluorescence intensity was measured with 515 nm excitation and 585 nm emission, and the images were analyzed using Image J software. To detect DNA damages from oxidative stress in the lesions, the aortic cryostat cross sections were incubated with 8-oxoguanine antibody (dilution factor of 1:200, #GTX41980 from GeneTex) at 37 °C for 1.5 h, and followed by anti-HRP second antibody and diaminobenzidine (to show the brown color).
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