AO staining and LTR staining were applied to assess the functional state of lysosomes in this study. After treatment with 2.5 μM Cd and/or 5 mM Tre for 12 h, cells grown on coverslips were loaded with 5 μg/ml AO at 37 °C for 30 min, rinsed two times with warm (37 °C) PBS and examined under confocal laser-scanning microscope (TCS SPE; Leica, Germany) with excitation at 488 nm. Green fluorescence (emission peak between 530 and 550 nm) and red fluorescence (emission peak at about 650 nm) were simultaneously collected by two separate windows. Meanwhile, cells were incubated with 100 nM LTR (diluted in DMEM-F12 medium) for 30 min under ideal growth conditions (37 °C, 5% CO2) to label the lysosomes. Then, slides were rapidly washed with warm PBS (37 °C) for three times, mounted as described above and observed under a laser-scanning confocal microscope (TCS SPE; Leica).
Tcs spe
Manufactured by Leica camera
Sourced in Germany, United States, United Kingdom, France
The Leica TCS SPE is a confocal laser scanning microscope designed for high-resolution imaging and analysis of biological samples. It provides precise control over the excitation light and detection of fluorescent signals, enabling detailed observation and study of cellular and subcellular structures.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (13 mentions)
Dm2500, by Leica camera (9 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (6 mentions)
Lsm 880, by Leica camera (5 mentions)
Acs apo 20 0.60 imm corr d, by Leica Microsystems (5 mentions)
Dmi8 base, by Leica camera (4 mentions)
Lsm 700, by Zeiss (4 mentions)
Tritc, by Southern Biotech (4 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (4 mentions)
Tcs sp5, by Leica camera (4 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (4 mentions)
Acs apo 63 1.30 oil cs 0 17 e objective, by Leica Microsystems (4 mentions)
Mitotracker red, by Thermo Fisher Scientific (4 mentions)
A0564, by Agilent Technologies (3 mentions)
A0565, by Agilent Technologies (3 mentions)
A0566, by Agilent Technologies (3 mentions)
Ab152, by Merck Group (3 mentions)
Ab92547, by Abcam (3 mentions)
Ab13970, by Abcam (3 mentions)
240 protocols using tcs spe
1
Lysosomal Functional Assessment by AO and LTR Staining
2
Visualization of ASC Inflammasome Specks
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The activation of the inflammasome requires ASC proteins to assemble into a large toroidal protein complex, which is termed “speck”24 (link). ASC-specks were visualized in HUVEC by indirect immunofluorescence as previously described7 (link). A primary polyclonal antibody against ASC (dilution 1:250; Molecular Probes) was used, followed by incubation with an appropriate Alexa 546-conjugated secondary antibody (dilution 1:100; Molecular Probes). Nuclei were counterstained with DAPI (5 µg/mL, Invitrogen) and cells were observed with a confocal microscopy (TCS SPE, Leica, Wetzlar, Germany). The percentage of specks was calculated as the percentage of cells displaying specks versus the total number of cell per field. Specks were first counted manually with a fluorescence microscope (Eclipse TE300; Nikon, Tokyo, Japan). In every preparation, we initially selected a central field and afterwards, following a counterclock radial pattern, eighth radius were traced and two fields were explored in each of them (total measurements, 17 fields per preparation at 100×). Representative images (63×) were obtained from every preparation with a confocal microscope (TCS SPE, Leica, Wetzlar, Germany).
3
Live/Dead Cell Viability Assay
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Cellular survival and proliferation are assessed with a Live/Dead viability kit, calcein AM (0.5 mL) and ethidium homodimer-1 (2.0 mL) were dissolved in 997.5 mL PBS, added to the samples, and incubated for 30 min while protected from light at 37 °C in a humidified atmosphere with 5% CO2. (EthD-1) (ThermoFisher Scientific #L3224, Karlsruhe, Germany) according to the manufacturer’s protocol. Afterwards, constructs are imaged in a fluorescence microscope (Smart Cell Imager PAULA, Leica Microsystems, Wetzlar, Germany) for green (live) and red (dead) spectra. Viability is screened at 1-, 6-, and 14-day post-printing with a Leica TCS SPE, Wetzlar, Germany, from MNCN-CSIC, Madrid. For the visualization of nuclear morphology and proliferation within the construct, cells were fixed with 4% w/v formaldehyde in PBS and stained with PBS containing 3 × 10−6 M, 4,6-diamidino-2-phenylindole DAPI (D1306, Invitrogen) and Actin (Alexa Fluor 488 phalloidin, A12379, Invitrogen) and by confocal microscopy using a confocal microscope (Leica TCS SPE, Wetzlar, Germany) from idiPAZ-Madrid.
4
Evaluating Antibiofilm Efficacy of E. coli Biofilms
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E. coli was cultured on various samples to form biofilms, and the antibiofilm performance is evaluated. The electrification groups were charged for 3 min every 8 h during bacteria cultivation. The films were seeded with E. coli (100 μl, 107 CFU ml−1) and incubated at 37 °C for 3 h. The samples with the bacteria were immersed into 2 ml of the lysogeny broth (LB) medium for 48 h to form biofilms, and the culture medium was refreshed every 12 h. Finally, the antibiofilm ability of different samples was evaluated by crystal violet staining and observing the 3-dimensional (3D) morphology. In crystal violet staining, the samples with biofilms were rinsed in PBS, immersed in the 0.1% crystal violet aqueous solution for 25 min, and gently rinsed with deionized water for 5 min. The bound crystal violet was dissolved in 1 ml of ethanol, and the eluate was analyzed on a multimode reader (BioTek, USA) to determine the absorbance at 590 nm [62 (link)]. In the 3D morphology observation, the films with biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (Thermo Fisher, USA) and observed under a confocal microscope (Leica, TCS SPE, Germany).
5
HEK-293 Cell Transfection with Nano-polyplex
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Briefly, HEK-293 cells were seeded on square glass placed at the bottom of six well plate at 30–40% confluency in complete DMEM-F12 medium. HEK-293 cells were then treated by nano-polyplex (pDNA-PDMAEA-GNRs). After 4 hours of incubation, the transfected cells were fixed with 10% formalin (Mojallali, Iran) and imaged by dark-field microscopy (Olympus, BX51TF, Japan). Likewise, for confocal microscopy the confluent cells were treated by SYBER Green-labeled nano-polyplex. Followed by 4 hours of incubation and fixing with 10% formalin, cells were stained with DAPI (Santa Cruz, sc-24941) for 5 minutes. The images were taken using a spectral confocal microscope (Leica TCS SPE, Germany).
6
Quantifying Immune Responses in Zebrafish
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1 and 4 dpi, transgenic zebrafish larvae infected with fluorescent M. marinum strains were mounted in 0.8–1% low melting point agarose (Sigma-Aldrich) and imaged on a Leica TCS SPE confocal on an inverted Leica DMi8 base and imaged using 20× or 40× objective lenses.
For quantification purposes, acquisition settings and area of imaging (in the caudal vein region) were kept the same across groups. Corrected total cell fluorescence was calculated for each immune-stained cell using Image J as previously described (18 (link), 21 (link)).
For quantification purposes, acquisition settings and area of imaging (in the caudal vein region) were kept the same across groups. Corrected total cell fluorescence was calculated for each immune-stained cell using Image J as previously described (18 (link), 21 (link)).
7
Immunohistochemistry of Formalin-Fixed Tissues
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For immunohistochemistry, formalin-fixed paraffin-embedded tissues were sliced into 4-mm-thick sections. Slides were deparafinized with xylene and heated for 15 min in citrate buffer (pH 6.0) using microwave. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol and then non-specific binding was blocked with 10% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween 20 (TBST) or mouse on mouse kit (VECTOR). The slides were incubated with primary antibodies and then incubated with appropriate secondary antibodies. Reacted antibodies were detected using ABC Elite kit (VECTOR) and diaminobenzidine (DAB) (VECTOR). Immunostaining of cultured cells was performed as described previously5 (link). Fluorescent images were obtained using TCS-SPE (Leica) and FLUOVIEW FV1200 (Olympus) confocal microscope systems.
8
Sciatic Nerve Injury and Analysis
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Mice were anesthetised with isofluorane, and the sciatic nerve was exposed and cut at the sciatic notch. The wound was closed using veterinary autoclips. The nerve distal to the cut was excised for analysis after 16 h and fixed with 4% PFA/PBS for 4 h at 4°C, then 15% sucrose/PBS overnight at 4°C, and then embedded in optimal cutting temperature compound. Transverse sciatic nerve cryosections (10 µm) were postfixed with 4% PFA/PBS for 10 min, blocked in 0.2% Triton X-100, 10% horse serum in PBS, and subsequently incubated with primary antibodies in blocking solution overnight at 4°C, followed by 1 h in secondary antibodies and DAPI to identify cell nuclei (1:30,000; Thermo Fisher Scientific). Samples were mounted with Fluoromount G. Imaging was performed at room temperature on a confocal microscope (TCS SPE; Leica) controlled with Leica confocal software. A 40× oil-immersion objective lens (40× HCX PL APO 1.25–0.75 OIL CS; Leica) was used for analysis. All confocal images represent the maximum projection from total stacks from 0–1 mm to the cut point, and six images per animal were counted using ImageJ software (National Institutes of Health).
9
Confocal Microscopy Reveals Bacterial Cell Lysis
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Confocal laser microscopy
was performed to determine the bacterial cell lysis caused by lead
inhibitor4l . The log-phase bacterial cells of E. coli were harvested by centrifugation at 3000
rpm for 15 min and washed twice with phosphate buffer saline. The
cell suspension was adjusted according to McFarland standards, and
then cells were exposed to MIC of4l for 3 h at 37 °C.
After exposure to test compound, the cells were again washed with
PBS and counterstained with DAPI for 30 min in the dark. The cells
were further washed to remove extra dye using PBS buffer solution.
The slides for treated and untreated samples were prepared, fixed
using poly(l -lysine), and observed under a confocal laser
scanning microscope Leica DMRE equipped with a confocal head TCS SPE
(Leica, Wetzlar, Germany) and a 60× water immersion objective
with a laser of 532 nm wavelength.
was performed to determine the bacterial cell lysis caused by lead
inhibitor
rpm for 15 min and washed twice with phosphate buffer saline. The
cell suspension was adjusted according to McFarland standards, and
then cells were exposed to MIC of
After exposure to test compound, the cells were again washed with
PBS and counterstained with DAPI for 30 min in the dark. The cells
were further washed to remove extra dye using PBS buffer solution.
The slides for treated and untreated samples were prepared, fixed
using poly(
scanning microscope Leica DMRE equipped with a confocal head TCS SPE
(Leica, Wetzlar, Germany) and a 60× water immersion objective
with a laser of 532 nm wavelength.
10
Subcellular Localization of Potato AREB/ABF/ABI5 Proteins
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The full-length coding sequences (CDS) of potato AREB/ABF/ABI5 genes with termination codon were amplified using potato cDNA as a template (refer to Table S1 for the primers used) and cloned into plasmid pH7LIC-N-eGFP with Exnase II (Vazyme, Nanjing, China) to produce the GFP-StAREB/ABF/ABI5s vectors for generating the GFP fused target protein in living cells. Subcellular localization of the GFP-StAREB/ABF/ABI5s was investigated at 60 h after infiltration by Agrobacterium tumefaciens GV3101 harboring the plasmid DNA with laser confocal fluorescence microscopy (Leica TCS-SPE, Wetzlar, Germany) in Nicotiana benthamiana epidermal cells.
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