Eclipse te 2000 u confocal microscope

IF experiments were performed with the antibody reported in Table S1, using an overnight incubation. Briefly, cells seeded onto glass slides and incubated with Cla, were fixed in 4% paraformaldehyde in PBS and permeabilized with 1% Triton X-100 in PBS. Anti-rabbit-Cy2 (1:1000) was used as secondary antibody, and nuclei were counterstained with Hoechst 33342 dye. Slides were examined with a Nikon Eclipse TE2000-U confocal microscope (Nikon, Tokyo, Japan).
To assess 11-NDB Cla binding by IF, HEK293 transfected with the different hERG1mutants were seeded on glass coverslips in 24-wells plates at a density of 3 × 10⁴ cells/well in complete medium. After 24 h, cells were treated for 30 min with 10 µM 11-NBD-clarithromycin, washed once with PBS at room temperature and fixed in 4% PFA for 15 min. Slides were imaged using a Nikon Eclipse TE2000-U confocal microscope (Nikon, Tokyo, Japan) (z-stacks steps = 0.5 µm, laser detection for 11-NBD-Cla = 450/35–515/30). Two types of analysis were then performed on exported confocal images: (i) mean 11-NBD-Cla fluorescence intensity was measured at the top focal plane of the image z-stack and normalized on selected cell areas using Fiji^{53 (link)} and (ii) mean fluorescence intensity was also quantified in single plane 2D confocal images and normalized on selected cell area using ImageJ.

Seven day-old seedlings were incubated in half MS medium containing DFPM at the indicated concentrations for 24 hours. Seedlings were stained with 7 μg/ml FDA and 5 μg/ml propidium iodide (Sigma, P4170) for 10 seconds. After a brief rinse with water, root cells were monitored using a Zeiss LSM 710 confocal microscope (Ex 488 nm/ Em 493–555 nm). Constant gain and pinhole values were used to observe fluorescence.
To quantify FDA fluorescence signals and calculate IC₅₀ values, seedlings exposed to DFPM with increasing concentrations from 0.5 to 10 μM were stained with 7 μg/ml FDA and observed using a Nikon Eclipse TE2000-U Confocal microscope (Ex 488 nm/ Em 500–550 nm). Fluorescence intensity was measured using Image J program [36 (link)] and IC₅₀ was measured using Graphpad Prism 6 program.

Confocal microscopy was used to determine antibody internalization in HER2-expressing N87 cells. Cells were grown in incubator overnight at 37 °C, 5% CO₂. Cells were washed with PBS and incubated with antibody (10 μg/ml) for 45 min at 4 °C to initiate binding. Unbound antibody was removed by washing with PBS then cells were returned to incubator in pre-warmed, complete RPMI medium. Cells were incubated for 24 hours then washed and fixed. To image internalization of unlabeled antibody, control samples were permeabilized and stained for 45 minutes with anti-human Fc fluorophore secondary antibody. Cells were washed and imaged on a Nikon Eclipse TE 2000-U confocal microscope.