1 × 105 cells were cultured on 14-mm cell climbing slices. At room temperature, cells at 80% confluency were subjected to fixation in 4% paraformaldehyde (PFA) for 20 min, followed by permeabilization with 0.3% Triton X-100 for 10 min and blockade with 10% goat serum in PBS for 1 h. Anti-CD147 (1:200, Abcam) primary antibody was incubated overnight at 4°C. The following day, CoraLite594-conjugated goat anti-rabbit IgG(H+L) (1:250, Proteintech) secondary antibody was added and incubation was observed for 1.5 h at room temperature. Cell climbing slices were sealed with mounting medium containing DAPI (Abcam, USA). Image acquisition was performed using the TCS SP8 confocal laser scanning microscopy (Leica, Italy).
Tcs sp8 confocal laser scanning microscopy
Manufactured by Leica
Sourced in Germany, Italy
The Leica TCS SP8 is a confocal laser scanning microscopy system. It is designed to provide high-resolution, three-dimensional imaging of biological samples. The system utilizes a laser light source and a series of optical components to scan the sample and collect fluorescence signals, enabling the visualization of cellular structures and processes.
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Lab products found in correlation
Coralite594 conjugated goat anti rabbit igg h l, by Proteintech (2 mentions)
Tcs sp5 laser scanning confocal microscopy, by Leica (2 mentions)
Pl apo cs 40 1.25 oil lens, by Leica (2 mentions)
Pl apo cs 63 1.40 oil lens, by Leica (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (1 mentions)
Anti cd147, by Abcam (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
Anti ldha, by Proteintech (1 mentions)
Coralite488 conjugated goat anti mouse igg h l, by Proteintech (1 mentions)
Anti β catenin, by Proteintech (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
Sc 20011, by Santa Cruz Biotechnology (1 mentions)
Sc 65882, by Santa Cruz Biotechnology (1 mentions)
Application suite x, by Leica (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Mouse anti his tag, by Thermo Fisher Scientific (1 mentions)
15 protocols using tcs sp8 confocal laser scanning microscopy
1
Quantitative Analysis of CD147 Expression
2
Immunofluorescence Staining of Cell Markers
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1 × 105 cells were seeded on cell climbing slices coated with poly-D-lysine in 24 well plates. Prepared cells were fixed in 4% PFA for 20 min, permeabilized with 0.5% Triton X-100 for 5 min, and blocked in 10% goat serum for 1 h at room temperature. The following primary antibodies were incubated overnight at 4 °C: anti-LDHA (1:250, Proteintech), anti-β-catenin (1:200, Proteintech), or anti-HIF-1α (1:200, Abcam; 1:100 Proteintech). Next day, cells were incubated with secondary antibodies for 1.5 h at room temperature: CoraLite594-conjugated goat anti-rabbit IgG(H + L) (1:250, Proteintech), CoraLite488-conjugated goat anti-mouse IgG(H + L) (1:250, Proteintech). Cell climbing slices were mounted on slides with mounting medium containing DAPI and photographed by a TCS SP8 confocal laser scanning microscopy (Leica, Italy) or an Axio Scope A1 microscope (Zeiss, Germany) at 400× magnification.
3
Intracellular Localization and Uptake of Nanoparticles
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To determine the intracellular localization, BV-2 cells were harvested with 1640 medium and seeded into a 20 mm glass bottom culture dish at a density of 3 × 105 cells/mL/well. After the cells were incubated at 37°C for 12 h, the medium was removed. Free DiO and NPs containing DiO at predefined concentrations were then introduced into each well containing 1 mL of medium. After administration for 6 h, cells were treated with Hoechst for staining in the nucleus and incubated at room temperature for 30 min to avoid light. Cells were rinsed twice with PBS buffer at pH 7.4 and fixed with 4% paraformaldehyde for 15 min at the same condition as Hoechst dying. Eventually, cells were imaged by Leica TCS SP8 Confocal Laser Scanning Microscopy (Berlin, Germany). The cellular uptake of NPs was quantitatively measured by flow cytometry. BV-2 cells in 24-well plates were treated with free DiO and NPs containing DiO for 1, 3, and 6 h. The medium was then removed, and cells were washed with PBS and trypsinized. The cells were collected and centrifuged at 1,000 rpm for 10 min, washed with PBS twice, and resuspended in 1 mL of PBS. Then, cell suspensions were filtered and finally subjected to flow cytometry equipped with an argon laser at 488 nm and analyzed with software through the fluorescence channel.
4
Quantifying CD71 and LAMP1 in Aspergillus-Infected Cells
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RCB2280 cells were cultured in 22 × 22mm coverslips and mixed with conidia (CI1698) at a multiplicity of ten and incubated for 6 h [25 (link)]. Following infection, cells were washed with 1X PBS, fixed with optimized fixation and permeabilization methods for CD71 (4% PFA and 0.1% triton X-100) and LAMP1 (MeOH-Acetone). After blocking, cells were incubated with mouse monoclonal antibodies of CD71 (transferrin receptor) (sc-65882, Santa Cruz Biotechnology, Texas, USA) or LAMP1 (sc-20011, Santa Cruz Biotechnology, Texas, USA) (1:50) for 1 h at room temperature. Finally, the cells were stained with goat anti-mouse IgG conjugated with dylight 550 (1:50) for 1 h at room temperature. Coverslips were mounted on glass slides using vectashield mounting medium containing DAPI and sealed with nail polish. The images were acquired using Leica TCS SP8 confocal laser scanning microscopy and analyzed using LAS AF Lite software.
5
Visualizing TRPV1 Receptor Binding
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VSMCs were seeded at an initial density of 5 × 104 cells/dish in 20-mm glass bottom dishes and incubated for 24 h before adding the test substance. Then fluorescein-conjugated CuS-TRPV1 and CuS was respectively cultured with VSMCs for 2 h at a concentration of 0.4 mg mL−1. In blocking group, excess amount of TRPV1 antibody (0.5 mg mL−1) was added to cells 30 min before fluorescein-conjugated CuS-TRPV1. In NIR group, cells were further irradiated by the 980 nm laser (5 W cm−2) for 30 cycles. After the wash step with PBS, the binding affinity of CuS-TRPV1 for TRPV1 on VSMCs was examined by TCS SP8 confocal laser scanning microscopy (Leica Co., Ltd. Germany) with 488 nm excitation. After confirming the cell focusing ranges from the bottom to top, z-height images were captured at 0.5, 2, 3.5, and 5 µm from the bottom and analyzed by Leica Application Suite X (Leica Co., Ltd. Germany) with a ×63 objective lens.
6
Visualizing Subcellular Localization of Plant Transcription Factors
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The N. benthamiana leaves were infiltrated with Agrobacterium cells containing 35S::VaRAV1-EGFP, 35S::VaERF1A-EGFP and 35S::VaCRF2-EGFP constructs, respectively, and the bacterial cells harboring the histone H2B-mCherry expression vector were co-infiltrated at a ratio of 1:1. The fluorescence was detected using Leica TCS SP8 confocal laser scanning microscopy. The experiment was repeated three times.
7
Protein-Protein Interaction Analysis using BiFc
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The bimolecular fluorescence complementation (BiFc) assay was performed to examine physical protein-protein interaction as previously described (Luo et al., 2017 (link); Pan et al., 2021 (link)). The plasmids expressing VN173, VC155, or fusion protein VN173-VPS25, VC155-3A were co-transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). At 24 h post-transfection, cells were pre-cultured at 4°C for 10 min. The fusion proteins in living cells were observed by Leica TCS SP8 confocal laser scanning microscopy.
8
Fluorescent Tagging of PcLAC4 and PcMYB44 in Plant Cells
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The coding regions of PcLAC4 and PcMYB44 without a stop codon were amplified from Conference pear cDNA and incorporated into the pCNY vector (Yang et al., 2018 (link)) fused in frame with the N‐terminus of the enhanced yellow fluorescent protein (eYFP) gene to generate fusion constructs designated 35S:PcMYB44‐eYFP and 35S:PcLAC4‐eYFP, respectively. These plasmids were transformed into Agrobacterium tumefaciens GV3101. For PcLAC4, onion epidermal cells were immersed in Agrobacterium suspension in MES‐MgCl2‐Acetosyringone (MMA) buffer (10 mM MES, 10 mM MgCl2, 150 μM AS, pH 5.6) at OD600 = 0.8 for 10 min, then transferred to MS medium for cultivation. Three days after transformation, cells were immersed in sucrose solution (0.3 g/mL) for plasmolysis before detection. For PcMYB44, 35S:PcMYB44‐eYFP and 35S‐eYFP plasmids were transiently co‐expressed with a nuclear marker (35S:H2B‐mCherry vector) (Martin et al., 2009 (link)) in N. benthamiana leaves. At 3 days postinfiltration, leaf tissues at the position of infiltration were collected for analysis. All samples were examined and fluorescence signals were measured using a TCS‐SP8 confocal laser scanning microscopy (Leica Microsystems).
9
Visualizing NF-κB Translocation in RAW264.7 Macrophages
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RAW264.7 macrophage cells were seeded on cover glass‐bottom dishes (Life Sciences) and pretreated in the absence or the presence of extracted gypenosides (200 μg/ml) for 1 hr and then stimulated with or without LPS (1 μg/ml) for 4 hr. Following the incubation, the cells were washed with PBS, fixed with cold 4% paraformaldehyde for 60 min and incubated with the anti‐NF‐κB p65 primary antibody (dilution 1:2,000) at 4°C overnight. Following the reaction, the cells were washed with PBS, treated with Alexa Fluor® 488 conjugate for 1 hr and then stained using DAPI (4 ng/ml) for 60 min at room temperature. After that, the cells were washed with PBS and Prolong Gold Anti‐fade Reagent® (Thermo Fisher Scientific, Inc.) was added to the slide. Lastly, the cells were visualized using a TCS SP8 confocal laser scanning microscopy (Leica Microsystems Inc.).
10
Immunofluorescence Staining of EV71 and Exosomes
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Rhabdomyosarcoma cells were seeded on 20-mm coverslips and infected with EV71. Then, the medium was removed and cells were washed with PBS, fixed with 4% formaldehyde for 30 min, and permeabilized with 0.2% Triton X-100 for 10 min at room temperature. After another PBS wash, cells were blocked in PBS containing 5% BSA for 1 h and incubated with mouse anti-EV71-3A antibody dilution (v/v = 1:200) and rabbit anti-VPS25 antibody dilution (v/v = 1:200) overnight at 4°C. Samples were incubated with FITC-conjugated goat anti-mouse and Cy3-conjugated goat anti-rabbit IgG dilution (v/v = 1:500) (ProteinTech Group, Wuhan, China) for 45 min at room temperature. For nuclei staining, 1 μg/ml DAPI in methanol was used and incubated with samples for 10 min at room temperature. After washing with PBS, the cells were observed by Leica TCS SP8 confocal laser scanning microscopy (Leica Microsystem, Wetzlar, Germany).
For the immunofluorescence staining of exosomes, exosomes were labeled with Dil membrane dye as previously described (Jiang Y. et al., 2020 (link)) and CoraLite®488-conjugated CD9 antibody dilution (v/v = 1:200). Labeled exosomes were washed with PBS and ultracentrifuged at 100,000 × g for 70 min at 4°C for three times. The samples were dropped on a glass slide and observed under Leica TCS SP8 confocal laser scanning microscopy with Lightning mode.
For the immunofluorescence staining of exosomes, exosomes were labeled with Dil membrane dye as previously described (Jiang Y. et al., 2020 (link)) and CoraLite®488-conjugated CD9 antibody dilution (v/v = 1:200). Labeled exosomes were washed with PBS and ultracentrifuged at 100,000 × g for 70 min at 4°C for three times. The samples were dropped on a glass slide and observed under Leica TCS SP8 confocal laser scanning microscopy with Lightning mode.
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