Cells were seeded onto 12-mm diameter glass coverslips coated with 0.1 mg/ml poly-l -lysine (Sigma-Aldrich, P5899), placed in 24-well plates, and transfected with plasmid DNAs using Lipofectamine 3000. After 24 h, the transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature or ice-cold 100% methanol for 15 min at –20°C, washed thrice with PBS, permeabilized/blocked with 5% FBS or 5% nonfat dry milk in PBS containing 0.3% Triton X-100 for 30 min at room temperature, and subsequently incubated with primary antibodies. The antibodies used for immunostaining are listed in Supplementary Table S3 . Following incubation, the cells were washed thrice with PBS, permeabilized/blocked with 5% FBS or 5% nonfat dry milk in PBS containing 0.3% and subsequently incubated with secondary antibodies conjugated with fluorescent dyes for 1 h at room temperature. The cells were washed thrice with PBS, permeabilized/blocked with 5% FBS or 5% nonfat dry milk in PBS containing 0.3%, stained with DAPI, mounted using Fluorescence Mounting Medium (Dako), and examined using a TCS SP5 laser-scanning confocal microscope (Leica Microsystems). To determine proportion of cells with centromeric ZFAT foci, cells were counted as ZFAT foci positive cells when >80% of the detected ZFAT foci were colocalized with CENP-A foci.
Tcs sp5 confocal laser scanning microscope
Manufactured by Leica Microsystems
Sourced in Germany, United States
The TCS SP5 is a confocal laser scanning microscope manufactured by Leica Microsystems. It is designed to provide high-resolution imaging of samples by scanning them with a focused laser beam and detecting the emitted fluorescence or reflected light.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica Microsystems (6 mentions)
Fluorescence mounting medium, by Agilent Technologies (4 mentions)
Dmi 6000 cs inverted microscope, by Leica camera (3 mentions)
Cellmasktm deep red, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Poly l lysine (pll), by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Dmi 6000 cs inverted microscope, by Leica Microsystems (2 mentions)
Las af software version 2, by Leica Microsystems (2 mentions)
Las af scan software, by Leica Microsystems (2 mentions)
Lab tek chambered coverglass, by Thermo Fisher Scientific (2 mentions)
Anti ipla2β, by Cayman Chemical (2 mentions)
Af488 conjugated goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Af594 conjugated goat anti rabbit, by Thermo Fisher Scientific (2 mentions)
μ slide vi0, by Ibidi (2 mentions)
Vectashield h 1000 medium, by Vector Laboratories (2 mentions)
Phosphate buffered saline (pbs), by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
73 protocols using tcs sp5 confocal laser scanning microscope
1
Centromeric Localization of ZFAT Protein
2
Immunofluorescence Staining of Human Met
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Immunofluorescence staining was performed as previously described using antibodies against human Met (1∶100) [21] (link). Hoechst 33342 (5 µg/ml; Beyotime Biotechnology) was used to visualize the nuclei. Stained specimens were visualized using a TCS-SP5 laser-scanning confocal microscope (Leica Microsystems, Germany).
3
Visualizing ZEB1 Expression in A549 Cells
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A549 cells were seeded on glass coverslips in an environment containing 5% CO2 in air at 37 °C and then incubated in 10 nM neutrophil elastase and 20 μM EGCG for an additional 24 h. The coverslips were gently washed in PBS, fixed in 3.7% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1% Triton X-100 for 20 min and blocked in PBS containing 3% BSA for 30 min. The fixed cells were incubated in an anti-human zinc finger E-box-binding protein-1 (ZEB-1) antibody (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C in a humidified chamber. Then, the cells were washed and incubated in the corresponding DyLight 488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at 37 °C. After washing in PBS, the cells were incubated in Hoechst 33342 (1 μg/ml, Sigma-Aldrich) for 5 min at room temperature. After several washes, the immunostained specimens were observed under a TCS-SP5 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
4
Confocal Microscopy for Biofilm Imaging
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An upright Leica Microsystems (Wetzlar, Germany) TCS SP5 laser-scanning confocal microscope was used (for data shown in Figure 1 D-I) with a 63× water immersion lens placed directly into the bulk fluid above biofilms grown on glass coupons or coverglass. Images were acquired through the Leica Application Suite Advanced Fluorescence software platform. Three-dimensional reconstructions of focal plane slices were generated and analyzed using IMARIS from Bitplane (Zürich, Switzerland; Figure 1 D-H) or Fiji [106 (link)]. An Andor spinning disk confocal system with a Nikon Eclipse Ti microscope (data shown in Figure 2 C,D,E,M) and Nikon A1R Spectral Confocal Microscope (Additional file 1 : Figure S2) were also used to image live biofilms within chamber slides.
5
Immunostaining of NHLRC2-HA and Caspase-FLAG in HEK293 Cells
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HEK293 cells seeded on to 12 mm diameter glass coverslips placed in 24-well plates were transfected with NHLRC2-HA expression vector alone or with myc-caspase-FLAG CS mutant vectors using Lipofectamine 3000 (Invitrogen). After 24 h, the transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min, washed with PBS three times, and then permeabilized/blocked with 0.3% Triton X-100/5% non-fat dry milk in PBS for 30 min. For immunostaining, the cells were first incubated with anti-HA (C29F4) and anti-FLAG (M2) antibodies for 1 h at room temperature. Both antibodies were diluted in 0.3% Triton X-100/5% non-fat dry milk in PBS at a dilution of 1:1,000. Following incubation, cells were washed with PBS three times and subsequently incubated with Alexa Fluor 555 conjugated anti-mouse IgG and Alexa Fluor 488 conjugated anti-rabbit IgG antibodies (both from Invitrogen) at a dilution of 1:1,000 for 1 h at room temperature. Cells were washed with PBS three times, mounted in Fluorescence Mounting Medium (Dako, Santa Clara, CA, USA), and examined using a TCS SP5 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
6
Intracellular Localization of C-NS-siRNA in 5-HT Neurons
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Intracellular C-NS-siRNA distribution in 5-HT neurons was examined by confocal microscopy using a Leica TCS SP5 laser scanning confocal microscope (Leica Microsystems Heidelberg GmbH, Manheim, Germany) equipped with a DMI6000 inverted microscope, blue diode (405 nm), Argon (458/476/488/496/514), diode-pumped solid state (561 nm) and HeNe (594/633nm) lasers. After i.n. administration with Alexa488 labeled C-NS-siRNA at 30 μg day−1 during 4 days, mice were killed and their brain were extracted and processed for immunofluorescence. Details are shown in Supplementary Information .
7
Immunofluorescent Imaging of AKAP79 and PKA RII
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SH-SY 5Y cells were plated on chamber coverslips. After drug treatment, the chamber coverslips were gently washed with PBS, fixed in 3.7% paraformaldehyde for 15 min, permeabilized in PBS containing 0.1% Triton X-100 for 5 min, and blocked in PBS containing 5% BSA for 30 min. The fixed cells were incubated with anti-AKAP79 and anti-PKA RII antibodies overnight at 4°C and incubated with the DyLight 488 and DyLight 594 conjugated secondary antibodies for 2 h. After PBS wash, nucleus was stained by Hoechst 33342 for 10 min. The images were recorded by TCS-SP5 laser scanning confocal microscope (Leica Microsystems, Germany).
8
Stable Expression of E-cadherin in HCT116 Cells
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The cDNA for human E-cadherin was inserted into a pMSCVpuro vector. Retroviruses were produced by transfection of the retroviral vectors as described previously (28) (link). HCT116 cells in six-well plates were infected with the viruses by centrifugation for 2 h at 32˚C and 2000 × g. Approximately 48 h after infection, the cells were treated with 2 μg/ml hygromycin (WAKO, Osaka, Japan) for one week to establish HCT116-derived cells stably expressing E-cadherin.
Immunofluorescence labeling. Immunofluorescence labeling using E-cadherin (Cell Signaling Technology) was performed as previously described (19, (link)32) (link). Cells were examined using a TCS SP5 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
Statistical analyses in cell culture experiments. Statistical analyses were performed using unpaired two-tailed Student's t-tests. All pvalues below 0.05 were considered to be statistically significant.
Immunofluorescence labeling. Immunofluorescence labeling using E-cadherin (Cell Signaling Technology) was performed as previously described (19, (link)32) (link). Cells were examined using a TCS SP5 laser-scanning confocal microscope (Leica Microsystems, Wetzlar, Germany).
Statistical analyses in cell culture experiments. Statistical analyses were performed using unpaired two-tailed Student's t-tests. All pvalues below 0.05 were considered to be statistically significant.
9
Immunofluorescence Staining of Transfected Cells
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Cells were seeded onto a 12-mm diameter glass coverslip coated with 0.1 mg/ml poly-L-lysine (Sigma-Aldrich, P5899) and placed into 24-well plates and subsequently transfected with plasmid DNA using Lipofectamine 3000 (Invitrogen). After 24 h, the transfected cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 15 min at room temperature, subsequently washed thrice with PBS, permeabilized, and blocked with 5% FBS or 1% nonfat dry milk in PBS containing 0.3% Triton X-100 for 30 min at room temperature and subsequently incubated with the primary antibodies. Antibodies used for immunostaining are detailed in Table S3 . Following incubation, the cells were washed thrice with PBS and subsequently incubated with secondary antibodies conjugated with fluorescent dyes, for 1 h at room temperature. Cells were then washed thrice with PBS, stained with DAPI, mounted using Fluorescence Mounting Medium (Dako), and viewed using a TCS SP5 laser-scanning confocal microscope (Leica Microsystems).
10
Visualizing Oxytocin Neuron MC4R Expression
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