Cells were seeded at a density of 3.0 × 105 cells/well in a 4-well chamber slide. After 24 h, the media were changed to serum-free media, containing the 100 ng/mL of BV. After 1 h, the cells were treated with 100 ng/mL of PgLPS (InvivoGen) for 7 h. The treated-cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were treated with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 2 min to permeabilize. Following permeabilization, the cells were blocked in PBS, containing 5% bovine serum albumin, at room temperature for 1 h. After blocking, the cells were incubated with diluted primary antibody overnight at 4 °C, and secondary antibody was performed at room temperature for 4 h. The nuclei were stained with Hoechst 33342 solution for 20 min. Slides were mounted by using fluorescence mounting medium (Dako, Santa Clara, CA, USA). Specimens were examined and photographed by the using confocal microscope system (Nikon, Tokyo, Japan). Antibodies used in the present study were the following: anti-TNF-α; anti-phospho-NF-κB (Santa Cruz); anti-mouse-, and anti-rabbit-biotinylated secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 555 (Thermo).
Confocal microscope system
Manufactured by Nikon
Sourced in Japan
The Nikon Confocal Microscope System is a high-resolution imaging tool designed to capture detailed, three-dimensional images of samples. It utilizes a focused laser beam and specialized optics to scan the sample point-by-point, allowing for the elimination of out-of-focus light and the creation of sharp, high-contrast images.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Anti phospho nf κb, by Santa Cruz Biotechnology (1 mentions)
Pg lps, by InvivoGen (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Microtome, by Leica camera (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Optimal cutting temperature compound, by Sakura Finetek (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dako fluorescence mounting medium, by Agilent Technologies (1 mentions)
Cyanine3 (cy3), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
6 protocols using confocal microscope system
1
Investigating TNF-α and NF-κB Signaling in LPS-treated Cells
2
Immunofluorescent Staining of Mouse Brain
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Briefly, mice were anesthetized with inhaled isoflurane and perfused transcardially with PBS. Brains were isolated rapidly. Tissues were fixed in 4% paraformaldehyde in 0.1 M PBS overnight, cryoprotected in 30% sucrose for at least 24 hrs at 4 °C, and then embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA). Frozen sections (35 μm) were prepared for immunofluorescent staining.
For immunofluorescent staining, sections were rinsed with PBS two times and incubated with blocking buffer (5% with 0.3% Triton X-100 in PBS) for 2 hrs at room temperature (RT). After incubation with with anti-GFAP (1:500) in blocking buffer for 24 hrs at at 4 °C. Then, the sections were incubated with secondary antibody for 2 hrs at RT and stained with DAPI to visualized nuclei before mounting.
All images were captured using a confocal microscope system by Nikon. 15–25 consecutive optical sections with 1 μm interval thickness at 20x magnification were captured for each z-stack image. Acquisition parameters, including photomultiplier gain and offset, were kept constant throughout each set of experiments.
For immunofluorescent staining, sections were rinsed with PBS two times and incubated with blocking buffer (5% with 0.3% Triton X-100 in PBS) for 2 hrs at room temperature (RT). After incubation with with anti-GFAP (1:500) in blocking buffer for 24 hrs at at 4 °C. Then, the sections were incubated with secondary antibody for 2 hrs at RT and stained with DAPI to visualized nuclei before mounting.
All images were captured using a confocal microscope system by Nikon. 15–25 consecutive optical sections with 1 μm interval thickness at 20x magnification were captured for each z-stack image. Acquisition parameters, including photomultiplier gain and offset, were kept constant throughout each set of experiments.
3
Immunostaining and Immunoblotting of Spinal Cord
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Mice were anesthetized and transcardially perfused with ice-cold PBS. The segments of lumbar spinal cord were quickly dissected out and cut into two parts: one for immunostaining and the other for immunoblotting. For immunostaining, the tissues were immediately perfused with ice-cold fixative solution containing 4% paraformaldehyde in PBS for 12 h at 4°C, followed by cryoprotection in 30% sucrose for 24 h at 4°C. After embedding in optimal cutting temperature compound (Sakura Finetek), the tissues were mounted on a microtome (Leica), and 10 µm frozen sections were prepared for indirect fluorescent immunostaining. The sections were incubated in blocking buffers (5% BSA, 0.3% Triton X-100 in PBS) for 1 h at room temperature, followed by overnight incubation with primary antibodies diluted in blocking buffer. The sections were subsequently stained with fluorescein isothiocyanate- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch) for 1 h at room temperature. Stained sections were mounted with mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories). All images were captured using a confocal microscope system (Nikon). NIH ImageJ software was used for quantitative analysis.
4
Immunofluorescence Imaging of Cell Lines
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Human fibroblasts, MEFs, and MCF-7 cells were mounted on glass slides. Cells were washed three times with PBS and then fixed with ice-cold 4% paraformaldehyde (PFA) for 15 min, followed by washing with PBS. Cells were permeabilized with 0.25% Triton X-100 for 15 min. Cells were then blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated with indicated primary antibodies overnight at 4°C. After washing three times with PBS, cells were incubated with the corresponding fluorescent secondary antibodies (Alexa Fluor–conjugated secondary antibody, 1:500; Life Technologies) for 1 hour at room temperature in the dark. The nucleus was stained with 4′,6-diamidino-2-phenylindole at room temperature for 5 min. Images were captured using a confocal microscope system (Nikon).
5
Quantifying Afferent Synapses in Noise-Exposed Cochleae
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In order to analyze the effect of noise exposure on primary afferent fiber (AF)/inner hair cell (IHC) synapses, immunofluorescence for synaptic ribbons was performed on surface preparations of the organ of Corti (5 cochleae/group). To identify and quantify afferent synapses, the specimens were incubated in a blocking solution [1% bovine serum albumin (BSA), 0.5% Triton X-100, and 10% Normal Goat Serum in phosphate buffered saline (PBS) 0.1 M] and, then, overnight at +4°C with a solution containing the following primary antibodies: mouse anti-CtBP2 (IgG1, 1:200; BD Transduction Laboratories) and mouse anti-GluA2 (IgG2a, GluR2/GluA2; 1:500; Millipore), according to published protocols (Paciello et al., 2018 (link)). Images of immuno-labeled specimens (40x) were taken by a confocal microscope system (Nikon). A paired synapse was identified as the one with co-localization of CtBP2 and GluA2 positive puncta and synaptic ribbons were then quantified using a compressed z-stack of each image using an image processing software (NIH ImageJ 1.43u, Image Processing and Analysis in Java). The total number of ribbons was divided by the number of IHCs to obtain a ribbon/IHC estimate. Analyses were performed on a total of 15 cells/groups.
6
Analyzing CD20 and CACNA1C Expression
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