Cells exposed to LPS with or without 2% of CO and 10 μM of CX for 24 h were studied. Cells were fixed with 4% paraformaldehyde and permeated with 0.1% Triton X-100. After being treated with primary p65 NF-κB antibody overnight, cells were then incubated for an additional hour with a secondary antibody (Alexa Fluor® 488). The cells were counterstained with Hoechst 33342 for five minutes and the final analysis was performed using a Nikon Eclipse Ni-U fluorescent microscope (Nikon Corporation, Tokyo, Japan).
Eclipse ni u fluorescent microscope
Manufactured by Nikon
Sourced in Japan
The Eclipse Ni-U is a fluorescent microscope designed for laboratory use. It features a motorized nosepiece, allowing for easy switching between multiple objective lenses. The microscope utilizes LED illumination and provides high-contrast, high-resolution imaging capabilities.
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9 protocols using eclipse ni u fluorescent microscope
1
NF-κB Activation Assay in LPS-Treated Cells
2
Immunofluorescent Detection of Tight Junction Proteins
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Antigen retrieval was achieved by immersing deparaffinized slide sections into 1X sodium citrate buffer (10 mM, pH 6, 0.05% Tween 20; Sigma, St. Louis, MO, USA) preheated to 95–100 °C. Slides were allowed to cool on a rocker in the citrate buffer for 20 min then washed 3 times in 1X PBS for 5 min each. Sections were blocked in 2% cold-water fish gelatin (Sigma, Inc., St. Louis, MO, USA) in 0.2% Triton for 1 h, then exposed to mouse anti-claudin-5 (1:100), mouse anti-claudin-6 (1:100) or rat anti-zonula occudens-1 (1:100) in blocking buffer for 24 h at 4 °C. Slides were washed 5 times in 1X PBS with 1% Tween 20 for 5 min each then treated with anti-mouse (1:400) or anti-rat (1:500) Alexa Fluor 594 and allowed to incubate in a dark chamber for 1 h at room temperature. Slides were further washed 4 times in 1X PBS. Negative controls were performed by omitting the primary antibody. Slides were mounted in FluoroshieldTM mounting solution with DAPI counterstain (Sigma, St. Louis, MO, USA) and viewed using a Nikon Eclipse Ni-U fluorescent microscope equipped with a digital Nikon DS-Ri2 camera and NIS Elements BR software, Version 5.21.01 (Nikon Instruments Inc., Nikon, Japan). Images were captured at the same exposure time to compare changes in expression over time.
3
CLE Impact on DNA Damage Evaluation
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To confirm the effect of CLE on DNA damage, the cells were seeded in 8-well cell culture slides for 3 days. RAW264.7 cells were supplemented with various concentrations of CLE (0.1–1000 µg/mL) or CX at 3.8 µg/mL, with or without 1 µg/mL of LPS, for 24 h at 37 °C in a humidified atmosphere with 5% CO2. The treated cells were fixed with 4% paraformaldehyde for 10 min and then treated with Hoechst 33,342 staining at 5 µg/mL for 10 min. The cells were then washed twice with PBS and observed under a Nikon Eclipse Ni-U fluorescent microscope (Nikon).
4
FFO Mitigates LPS-Induced DNA Damage
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To confirm the effect of FFO on LPS-induced DNA damage, RAW264.7 cells were seeded into 8-well cell culture slides and treated with different concentrations of FFO [0.125-2% in 0.5% propylene glycol (v/v)] with or without LPS (1 µg/ml) for 24 h at 37˚C in a humidified atmosphere with 5% CO2. Treated cells were fixed with 4% paraformaldehyde for 10 min at room temperature and subsequently stained with Hoechst 33342 (5 µg/ml) for 10 min at room temperature. Cells were washed twice with PBS and observed under a Nikon Eclipse Ni-U fluorescent microscope (original magnification, x40; Nikon Corporation).
5
Quantifying Dopaminergic Neurons in Flies
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DA neurons were counted using a Nikon Eclipse Ni-U fluorescent microscope equipped with a 20× objective. DA neurons located in the PPL1, PPM1/2, and PPM3 were counted for both hemispheres for each brain sample. A minimum of 10 brains were analyzed for each genotype and sex in every experiment. Male and female samples were analyzed separately, and only combined if there were no statistically significant differences between sexes. For mutants of genes located on the X chromosome, hemizygous males were analyzed. Since no differences were observed between sexes in any of our measurements, the data presented represents combined results. Experiments were performed in triplicates and were scored blindly with regard to genotype or condition. Average values for each measurement are listed in Supplementary Table S3.
6
Immunofluorescent analysis of muscle fibers
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Gastrocnemius muscle samples were fixed in 4% paraformaldehyde, embedded with paraffin, and sliced into 4 μm sections. Paraffin sections were dewaxed in xylene and rehydrated in graded alcohol. Sections were microwave-heated to 95°C and kept for 20 min in Tris-EDTA (pH 9.0) buffer for the antigen retrieval step. Incubate sections in 5% BSA, 5% goat serum, 5% donkey serum in PBST at room temperature for 1 h to block unspecific binding of the antibodies. Antibodies were used as follows: MYH7 (Abcam, ab11083) and MYH4 (Abcam, ab91506) as the primary antibody; Goat Anti-Mouse IgG H&L (Alexa Fluor 594) (ab150116) and Donkey Anti-Rabbit IgG H&L (Alexa Fluor 488) (ab150073) as the secondary antibody. Fluorescent images were taken with ECLIPSE Ni-U fluorescent microscope (Nikon, Japan) with Digital Sight 10 imaging system and NIS-Elements software. The number and area of muscle fibers in each image were counted using ImageJ software.47 (link)
7
Microscopic Visualization of Dauer and Non-Dauer Nematodes
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Dauer and non-dauer animals were placed on 2% agarose pads on glass slides, immobilized with 1 mM or 20 mM levamisole respectively. The preparation was covered with glass coverslips and subsequently observed under a 40x or 60x oil objective in an upright Nikon Eclipse Ni-U fluorescent microscope. For high resolution images in Figs 3I and 4G , we used a Leica TCS SP5X microscope.
8
Apoptosis Analysis by AO/EB Staining
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The dual acridine orange/ethidium bromide (AO/EB) staining method was used for the morphological analysis of apoptosis. The cells were seeded on coverslips at a density of 8·105 cells/slide and incubated at 37 °C for 48 h. Then, the medium was removed and solutions of 5 s , 5 u , 7 f and DOX at the IC50 concentration were added. After 24 h, the cells were washed with PBS, and then the cells were incubated in a staining solution (100 μg/mL AO and 100 μg/mL EB in PBS) (Sigma-Aldrich, St. Louis, MO, USA) for 3–4 min. After staining, the coverslips were washed with PBS and fixed with 3.7% paraformaldehyde for 15 min. Observations and photography were performed with a Nikon Eclipse Ni-U fluorescent microscope equipped with the sets of filters for AO and EB (excitation filter 450–490, barrier filter BA520).
9
Quantifying Dopaminergic Neurons in Drosophila
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DA neurons were counted using a Nikon Eclipse Ni-U fluorescent microscope equipped with a 20x objective. DA neurons of interest located in the protocerebral posterior lateral-1 (PPL1), Protocerebral posterior medial 1 and 2 (PPM1/2), and Protocerebral posterior medial 3 (PPM3) clusters were counted for both hemispheres within each individual brain sample. Brains were analyzed by genotype and sex for each experiment. A minimum of ten brains were analyzed for each group. Male and female brains were analyzed separately and then combined if there was no statistical difference between them. For mutants of CG42339 located on the X chromosome, only hemizygous males were analyzed. Experiments were performed in triplicate and were scored blindly with regard to genotype and condition.
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