The VK2/E6E7 cells were grown on glass coverslips, fixed with 4% paraformaldehyde for 40 min, and then permeabilized with 0.1% Triton X-100 for 20 min at room temperature (RT). Cells were then blocked with 3% bovine serum albumin (Sigma-Aldrich) for 2 h to block non-specific antibody binding and then incubated overnight at 4 °C with antibody against HBD-1 (Abcam, Cambridge, UK). After washing, cells were incubated with FITC-conjugated anti-mouse IgG (1:1000 dilution; Sigma-Aldrich) for 1 h at RT, and DNA was counterstained with propidium iodide (Sigma-Aldrich). Coverslips were mounted with Fluorescence Mounting Medium (DAKO). Fluorescence was detected by confocal laser microscopy using a Zeiss LSM510 instrument (Zeiss, Jena, Germany).
Lsm 510 instrument
Manufactured by Zeiss
Sourced in Germany
The Zeiss LSM 510 is a high-performance confocal laser scanning microscope. It is designed to provide researchers with advanced imaging capabilities for a wide range of applications. The instrument utilizes a series of precision-engineered optical components and sophisticated control systems to capture high-resolution, three-dimensional images of biological samples and other materials.
Automatically generated - may contain errors
Lab products found in correlation
Aim image examiner software, by Zeiss (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Fitc conjugated anti mouse igg, by Merck Group (1 mentions)
Malvern nano zs zetasizer, by Malvern Panalytical (1 mentions)
Fixation permeabilization buffer, by BD (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
4 protocols using lsm 510 instrument
1
Immunofluorescence Analysis of HBD-1 Expression
2
Protein Localization in Cell Lines
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U87MG-EGFP and CHO-EGFP cells were grown in collagen-coated 35 mm glass-base dishes (Asahi techno glass, Tokyo, Japan) for 24 h in growth medium. Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde at 4°C for 30 min. The cells were then incubated with the purified GCR or GC fusion proteins (2 μg) at 37°C for 1 h and washed two times with PBS. Confocal scanning laser microscopy was performed with a Zeiss LSM 510 instrument (Carl Zeiss, Oberkochen, Germany) with a 40x objective. The images were processed using Aim Image Examiner software (Carl Zeiss, Oberkochen, Germany).
3
Characterization of Multilayered Nanoparticles
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Laser
Doppler electrophoresis measurements were performed using a Malvern
Nano-ZS Zetasizer (Malvern Instruments Ltd, Worcestershire, UK). The
stability property of prepared NPs was investigated by measuring the
change in NP size in 10% human serum (v/v) and 0.01 M PBS (pH 7.4)
at room temperature under continuous stirring. The multilayered structure
of the NPs was characterized by TEM (JEOL JEM 1400 instrument, JEOL
Ltd., Japan) at a voltage of 120 kV and CLSM (Zeiss LSM 510 instrument,
Carl Zeiss, Germany). A zetasizer test was completed using samples
that were freshly prepared before use by dispersing the NPs in ultrapure
water. To image the NPs by negative TEM staining, the NPs were dissolved
in 0.01 M of pH 7.4 PBS buffer and were negatively stained according
to a standard procedure.65 (link) Fluorescent
Ch-MLNPs were imaged by CLSM, in which the PLGA, liposome, and chitosan
layers were labeled by DOX (red), NBD (green), and Alexa Fluor 350
(blue), respectively.
Doppler electrophoresis measurements were performed using a Malvern
Nano-ZS Zetasizer (Malvern Instruments Ltd, Worcestershire, UK). The
stability property of prepared NPs was investigated by measuring the
change in NP size in 10% human serum (v/v) and 0.01 M PBS (pH 7.4)
at room temperature under continuous stirring. The multilayered structure
of the NPs was characterized by TEM (JEOL JEM 1400 instrument, JEOL
Ltd., Japan) at a voltage of 120 kV and CLSM (Zeiss LSM 510 instrument,
Carl Zeiss, Germany). A zetasizer test was completed using samples
that were freshly prepared before use by dispersing the NPs in ultrapure
water. To image the NPs by negative TEM staining, the NPs were dissolved
in 0.01 M of pH 7.4 PBS buffer and were negatively stained according
to a standard procedure.65 (link) Fluorescent
Ch-MLNPs were imaged by CLSM, in which the PLGA, liposome, and chitosan
layers were labeled by DOX (red), NBD (green), and Alexa Fluor 350
(blue), respectively.
4
Confocal Microscopy Analysis of NK Cell Markers
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For confocal microscopic analysis, NK92-MI and NKF cells were seeded at 2 × 104 cells per chamber on Laboratory-Tek German borosilicate cover glass with eight chambers (Nunc, Rochester, NY, USA) and incubated for 24 h in growth medium. The cells were washed twice with 200 μL PBS. Subsequently, the cells were fixed and permeabilized with Fixation/Permeabilization buffer (BD Bioscience, San Jose, CA, USA) for 30 min at 4°C. The cells were then washed with 200 μL 1× BD wash buffer and incubated with phycoerythrin (PE)-conjugated anti-mouse CD90.1 (BD Bioscience) antibody at room temperature for 1 h followed by three washes with 200 μL 1× BD wash buffer. The slides were mounted with Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) and covered with glass cover slips. Confocal scanning laser microscopy was performed using a Zeiss LSM 510 instrument (Carl Zeiss, Oberkochen, Germany) with a 40× oil objective, as indicated. The images were processed using Aim Image Examiner software (Carl Zeiss).
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