To confirm the effect of ASI on nuclear translocation of NF κB, ICC staining was conducted. BV-2 cells were pre-treated with or without ASI (50 μM) for 2 h prior to the stimulation of LPS (200 ng/ml). One hour later, the cells were washed with PBS twice followed by fixation with 4% PFA for 10 min. Afterwards, they were permeablized and blocked with 5% normal donkey serum in PBS containing 0.1% Triton X-100. Half an hour later, the cells were incubated with phospho-NF κB p65 antibody at 4 °C overnight. After thoroughly washed with PBS, they were further incubated with Alexa-594 conjugated secondary antibody at room temperature for 1 h. Thereafter, the cells were mounted with Vector mounting medium supplemented with DAPI after washed with PBS. The fluorescence of the cells was visualized under Olympus DX81 fluorescent microscope.
Dx81 fluorescent microscope
Manufactured by Olympus
Sourced in Japan
The DX81 is a fluorescent microscope manufactured by Olympus. It is designed to visualize and analyze fluorescently-labeled samples. The microscope features high-quality optics and a range of illumination and filter options to support various fluorescence imaging techniques.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using dx81 fluorescent microscope
1
NF-κB Nuclear Translocation Assay
2
LPS-Induced NF-κB Activation in BV-2 Cells
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The BV-2 cells were seeded in 8 chamber slides (BD Biosciences, San Diego, CA, USA) at a density of 2x10 5 cells/ml. Tweety-four hours later, the cells were treated with or without ISO I (100 µM) for 2 h followed by stimulation with LPS (200 ng/ml) for 20 h. Thereafter, they were washed with phosphate-buffered saline (PBS), fixed with 4% PFA, permeabilized and blocked in PBS with 5% normal donkey serum and 0.1% Triton X-100. Half an hour later, cells were incubated with p-NF-κB p65 antibody overnight at 4̊C. After washing in PBS, they were further incubated with Alexa-594 conjugated secondary antibody at room temperature for 1 h. At last, cells were sealed in medium with DAPI. Fluorescence images of the cells were acquired using an Olympus DX81 fluorescent microscope (Olympus, Tokyo, Japan).
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