Cells were transfected with arrestin3-GFP along with D2Arr or D2G. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 20°C. The cells were subsequently mounted on slides using Vectashield (Vector Laboratories; Burlingame, CA, USA) and imaged using a laser scanning confocal microscope (TCS SP5/AOBS/Tandem; Leica, Jena, Germany). The images were analyzed using the Fiji version of the image processing software ImageJ and colocalization was analyzed based on Pearson’s correlation coefficient (γ value) (Min et al., 2023 (link)).
Tcs sp5 aobs tandem
Manufactured by Leica
Sourced in Germany
The Leica TCS SP5 AOBS/Tandem is a laser scanning confocal microscope system. It features an Acousto-Optical Beam Splitter (AOBS) and a Tandem scanning system. The AOBS allows for flexible and efficient control of the excitation laser lines, while the Tandem scanning system enables fast and high-resolution image acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (2 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (2 mentions)
Las af software, by Leica (2 mentions)
Las x program, by Leica (1 mentions)
Prolong antifade reagent, by Thermo Fisher Scientific (1 mentions)
Eclipse ti e, by Oxford Instruments (1 mentions)
Rm2235, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Evos fl auto 2, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Propidium iodine, by Thermo Fisher Scientific (1 mentions)
Bay11 7082, by Merck Group (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Alexa fluor 488 conjugated anti rabbit igg antibody, by Thermo Fisher Scientific (1 mentions)
24 protocols using tcs sp5 aobs tandem
1
Imaging Arrestin3-GFP Colocalization
2
Biofilm Visualization and Viability Assay
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Each of the four silicone sheets from four different treatment groups (I, II, III, and IV) intended for imaging was subjected to a LIVE/DEAD BacLight™ Bacterial Viability Kit (ThermoFisher Scientific Co., Waltham, MA, USA) and staining kits for CRPA. In this study, we omitted the groups E and F, because SEM and AFM showed no effect of AlgL-only treatment groups. Directly after the silicone sheets were removed, they were stained for 15 min in the dark at room temperature with both the live/dead viability stains containing SYTO 9 dye (3.34 mM) and propidium iodide (20 mM). A series of about 20 images was generated for each biofilm on the silicone sheets using confocal laser scanning microscopy (laser scanning confocal microscope, TCS SP5/AOBS/Tandem, Leica, Germany).
3
Quantifying Nuclei Morphology and Protein Levels
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The fibroblasts were plated onto poly-L-lysine-coated coverslips, serum-deprived for 24 h, fixed in 4% PFA, and immunolabeled as described previously (25 (link), 28 (link)).
To count the nuclei with irregular morphology, the samples were immunostained using rabbit polyclonal anti-LB1 or goat anti-LB and counterstained with Hoechst-33342. The confocal optical sectioning was performed at RT using a Leica TCS SP5 AOBS TANDEM inverted confocal microscope that was equipped with a HCX PL APO 40 ×1.25 Oil and a HCX PL APO λ blue 63 ×1.4 NA Oil objective lenses. The nuclei were scored as normal (oval or spherical shape) or as misshapen (irregular contour, the presence of blebs and membrane invaginations) by an operator who was blind to the nature of the samples. The count of RNA polymerase II-positive nuclei was performed on confocal images of blind-coded samples immunostained for RNA polymerase II by an experienced researcher. To quantify protein levels, z-stack confocal images of ADLD and CTR nuclei were acquired alongside in the same confocal session using fixed laser and scanning settings. With the use of maximal projection confocal images, nuclei were selected and the protein immunoreactive area was measured using the Leica LAS and the NIH ImageJ softwares (26 ). Data are normalized on the area covered by DNA staining with the Hoechst-33342 dye.
To count the nuclei with irregular morphology, the samples were immunostained using rabbit polyclonal anti-LB1 or goat anti-LB and counterstained with Hoechst-33342. The confocal optical sectioning was performed at RT using a Leica TCS SP5 AOBS TANDEM inverted confocal microscope that was equipped with a HCX PL APO 40 ×1.25 Oil and a HCX PL APO λ blue 63 ×1.4 NA Oil objective lenses. The nuclei were scored as normal (oval or spherical shape) or as misshapen (irregular contour, the presence of blebs and membrane invaginations) by an operator who was blind to the nature of the samples. The count of RNA polymerase II-positive nuclei was performed on confocal images of blind-coded samples immunostained for RNA polymerase II by an experienced researcher. To quantify protein levels, z-stack confocal images of ADLD and CTR nuclei were acquired alongside in the same confocal session using fixed laser and scanning settings. With the use of maximal projection confocal images, nuclei were selected and the protein immunoreactive area was measured using the Leica LAS and the NIH ImageJ softwares (26 ). Data are normalized on the area covered by DNA staining with the Hoechst-33342 dye.
4
Visualizing Plant Cell Plasmolysis
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The fluorescence signal from the reporter protein and the FM4-64 dye (70021, Biotium, Fremont, CA, USA) was observed using confocal laser scanning microscopy (TCS SP5 AOBS Tandem, Leica, Germany). Four-day-old seedlings were stained with 2 μM FM4-64 for 5 min, washed with distilled water for 5 min, and plasmolyzed by 0.2 M NaCl treatment for 1 min. YFP and RFP signals were detected using 514/>530 and 543/560–615 nm excitation/emission filter sets, respectively. Fluorescence images were digitized using the Leica LAS X program. The images were acquired at the Korea Basic Science Institute, Gwangju, Korea.
5
Investigating Nuclear Localization of AsphyA Kinase Mutants
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To investigate nuclear localization of the kinase mutants (K411R and T418V), transgenic phyA-201 plants expressing eGFP-fused AsphyA constructs were used. Four-day-old dark-grown seedlings were either kept in the dark or exposed to white light (100 μmol⋅m–2⋅s–1) for 5 min before the subcellular localization analysis. For analyzing GFP fluorescence, seedlings were transferred onto a microscope slide, covered with a cover slip, and observed using a laser scanning confocal microscope (Leica TCS SP5 AOBS/Tandem) at the Gwangju Center of Korea Basic Science Institute (KBSI).
6
Subcellular Localization of PgCYP736A12
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For subcellular localization of PgCYP736A12-ECFP, the fluorescence emitted from reporter proteins was observed by confocal laser scanning microscopy (TCS SP5 AOBS/Tandem; Leica Germany). ECFP was detected using 436/20 and 480/40 nm excitation/emission filter sets. Images were acquired at the Korea Basic Science Institute, Gwangju.
7
Immunofluorescence Microscopy Imaging Protocol
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Cells on coverslips were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min and permeabilised with 0.05% Triton X-100 for 10 min. After 30 min blocking with 1% BSA, cells were incubated for 1 hour with the primary antibody in a wet chamber, washed three times with PBS-T and incubated for 45 min with the secondary antibody. Finally, the cells were washed three times with PBS-T and mounted in Prolong anti-fade reagent (Life Technologies).
Wide-field images were acquired by Leica DM8000. Confocal images were acquired using Leica TCS SP5 AOBS TANDEM. In both cases imaging was performed with 63× immersion oil objective lens, NA 1.4 and a LAS AF software. Superresolution images were acquired using microscope Nikon ECLIPSE Ti-E equipped with an Andor iXon3 897 EMCCD camera and objective CFI SR Apochromat TIRF 100x/1.49 oil. Software NIS-Elements AR 4.20.01 and NIS Elements AR 4.30 were used for capturing and analysis of the images. Resolution obtained at the time of measurements was 120 nm.
Wide-field images were acquired by Leica DM8000. Confocal images were acquired using Leica TCS SP5 AOBS TANDEM. In both cases imaging was performed with 63× immersion oil objective lens, NA 1.4 and a LAS AF software. Superresolution images were acquired using microscope Nikon ECLIPSE Ti-E equipped with an Andor iXon3 897 EMCCD camera and objective CFI SR Apochromat TIRF 100x/1.49 oil. Software NIS-Elements AR 4.20.01 and NIS Elements AR 4.30 were used for capturing and analysis of the images. Resolution obtained at the time of measurements was 120 nm.
8
Poplar Stem Lignin Imaging
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Using a rotary microtome (RM2235, Leica, Germany), the stem of 24-week-old poplar was sectioned in a transverse axis to a thickness of 70 μm. Autofluorescence of the lignin was then observed by confocal laser scanning microscopy (TCS SP5 AOBS/Tandem, Leica Germany) under ultraviolet (UV) light. The images were acquired at the Korea Basic Science Institute, Gwangju. Korea.
9
Immunostaining and Confocal Imaging of Arrestin3
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Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 20 °C, washed three times with PBS, and then incubated with blocking buffer (1% FBS and 1% BSA in PBS) for 1 h at 20 °C. The cells were then incubated with antibodies against arrestin3 or HA (1:1000 dilution) for 1 h at 20 °C, followed by washing with PBS and incubation with the secondary antibodies (Alexa 555-conjugated antibodies at 1:500 dilution). The immunostained cells were subsequently mounted on slides using Vectashield (Vector Laboratories; Burlingame, CA, USA) and imaged using a laser scanning confocal microscope (TCS SP5/AOBS/Tandem; Leica, Jena, Germany). The images were analyzed using the Fiji version of the image processing software ImageJ and colocalization was analyzed based on Pearson's correlation coefficient (γ value).
10
Immunofluorescence Imaging of OSCC Cells
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Immunofluorescence was performed as described previously [53 (link)]. Briefly, OSCC cells were blocked with 3% BSA at room temperature for 1 h and then probed overnight with primary antibodies at 4 °C. After washing three times, the cells were probed with secondary antibodies (Invitrogen, Carlsbad, CA, USA) for 2 h at room temperature. After washing three times, the cells were stained with DAPI (Sigma-Aldrich) for 5 min. Immunofluorescence images were captured using a fluorescence and laser scanning confocal microscope (Leica TCS SP5 AOBS/Tandem) at the Gwangju Center of Korea Basic Science Institute (KBSI).
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