Folic acid (FA), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), 3,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and N-acetyl cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). Primary and secondary antibodies as well as DAPI (4′,6-diamidino-2-phenylindole) were purchased from Cell Signaling Technology.
Dapi 4 6 diamidino 2 phenylindole
Manufactured by Cell Signaling Technology
Sourced in United States
DAPI (4′,6-diamidino-2-phenylindole) is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in fluorescence microscopy to visualize and stain cell nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Aqua poly mount, by Agilent Technologies (2 mentions)
Fluorescent conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
Primary and secondary antibodies, by Cell Signaling Technology (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Spinsr10 confocal system, by Olympus (1 mentions)
F7250, by Merck Group (1 mentions)
Cellsens dimension, by Olympus (1 mentions)
Succinate assay kit colorimetric, by Abcam (1 mentions)
Ponceau s solution, by Merck Group (1 mentions)
Recombinant murine srank ligand rankl, by Thermo Fisher Scientific (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Macrophage colony stimulating factor m csf, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Merck Group (1 mentions)
880 laser scanning confocal microscope, by Zeiss (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Axio imager lsm 800, by Zeiss (1 mentions)
Paraformaldehyde (pfa), by Beyotime (1 mentions)
Cm1950, by Leica (1 mentions)
9 protocols using dapi 4 6 diamidino 2 phenylindole
1
Oxidative Stress and Cellular Assays
2
Amoxapine Modulates BCG Infection in LC3-GFP Cells
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RAW LC3-GFP cells were seeded on a sterilized glass coverslip 1 day before infection. Cells were infected with tdTomato-labeled BCG Danish or unlabeled BCG Danish (MOI = 10), then treated with 10 μM Amoxapine at the indicated concentrations for 24 h. Cells were rinsed once with PBS and fixed in 4% formalin/PBS for 10 min at room temperature. Cells were then washed three times with PBS, and the coverslips were mounted on slides with a prolonged gold antifade reagent and DAPI (4′,6-diamidino-2-phenylindole; Cell Signaling Technologies). Images were taken using an A1 Nikon confocal microscope (Nikon, Tokyo, Japan) and analyzed with NIS Elements (Nikon).
3
Immunofluorescence Imaging of GAS Infection
4
Succinate Signaling Pathway Regulation
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Metformin was from Calbiochem (Darmstadt, Germany). Dulbecco's Phosphate-Buffered Saline, Succinate (Succinic acid) and Ponceau S solution were from Sigma-Aldrich (St Louis, MO, USA).The Succinate Colorimetric Assay Kit (catalogue number: K649) was from BioVision (Milpitas, CA, USA). Ten per cent buffered formalin was from Fisher Scientific (Hampton, NH, USA). Recombinant murine sRANK ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) were from PeproTech (Rocky Hill, NJ, USA). Antibodies against p50, p65, β actin, DAPI (4′,6-diamidino-2-phenylindole) and Histone H3 were purchased from Cell Signalling (Danvers, MA, USA), SUCNR1 was from Novus Biologicals (Littleton, CO, USA). The SUCNR1 antagonist 4c (4c) was synthesized by Shenyang Wuhe BioTech Co. (Liaoning, China) based on the previous study34 (link). Details on the (monoclonal) antibody target, clone name, source and dilution used for all antibody based methods can be found in the Supplementary Table 3 .
5
Immunofluorescence Staining of Tight Junction Proteins
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Frozen sections were cut at 10 μm, and mounted on the slides. Antigen was retrieved by incubating the slides in 10 mM sodium citrate (pH 6) at 90 °C–100 °C. The nonspecific background was blocked by incubation with 5% bovine serum albumin plus 5% newborn bovine serum in PBS for 30 min at room temperature. The sections were incubated with rabbit polyclonal antibody against ZO-1 (Abcam, 1:200 dilution), rabbit polyclonal antibodies against occluding (Abcam, 1:200 dilution), rabbit polyclonal antibody against claudin-1 (Abcam, 1:200 dilution) and p65 (Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C overnight. The sections were probed with FITC-conjugated or PE-conjugated (only for p65) secondary IgG antibodies. The nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole, Cell Signaling Technology, Beverly, MA, USA). Slides incubated without any primary antibody were used as negative controls. Confocal analysis was performed with a confocal scanning microscope (BX25, Olympus, Tokyo, Japan).
6
Immunostaining of LPS-stimulated iBMDMs
7
Confocal Microscopy of TNF-α Induced NF-κB Translocation
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The confocal microscopy experiments were carried out as described previously (48 (link)–50 (link)). Briefly, HeLa cells were plated on 24-well plates in DMEM added with 10% FBS at a density of 2 × 105 cells per well overnight before transfection, and then cells were transfected with 500 ng of the indicated plasmids. Twenty-four hours posttransfection, cells were treated with 10 ng/mL of recombinant human TNF-α or mock treated for 30 min, and then the cells were fixed with 4% paraformaldehyde (Beyotime Biotechnology, Shanghai, China) for 30 min at 37°C and incubated in 0.2% Triton X-100 (Beyotime Biotechnology) for 30 min. After that, the cells were probed with the indicated Abs (anti-p65, anti-p50, or anti-HA) for 1.5 h at room temperature, followed by incubation with FITC-conjugated donkey anti–mouse IgG or Cy5-conjugated goat anti–rabbit IgG for 1 h. Finally, the cells were stained with DAPI (4’6-diamidino-2-phe-nylindole) (Cell Signaling Technology, Inc., Danvers, MA, United States) for 3 to 5 min. Images were obtained with a confocal microscope (Axio-Imager-LSM-800; Carl Zeiss, Oberkochen, Germany) using 400 × oil-immersion objective. Each image represented a vast majority of the cells with similar subcellular distribution.
8
Perfusion, Fixation, and Staining for Brain Analysis
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The rat was deeply anesthetized with 1% pentobarbital sodium (50 mg/kg, i.p.) and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde (PFA, in 0.1 M phosphate buffer, pH 7.4). The isolated brain was post-fixed in 4% PFA for at least 12 h, and dehydrated in 20% and 30% sucrose solutions in turn. The fixed brain was cut coronally at 40 µm on a cryostat microtome (CM1950, Leica, Germany).
Recording sites were identified by visual examination of electrolytic lesions, which were induced immediately before perfusion by passing current (2 mA, 15 s) through the electrode [20 (link)]. The brain slices were stained with Nissl solution and photographed under a light microscope (Leica DMI 4000B, Wetzlar, Germany).
Virus infection was validated after behavioral tests. Brain slices were mounted after incubation with a DNA-specific fluorescent probe (DAPI: 4′,6-diamidino-2-phenylindole, 1:1,000, Cell Signaling Technology). Fluorescence images were acquired using a laser scanning confocal microscope (model FV1000, Olympus Co., Ltd.)
Recording sites were identified by visual examination of electrolytic lesions, which were induced immediately before perfusion by passing current (2 mA, 15 s) through the electrode [20 (link)]. The brain slices were stained with Nissl solution and photographed under a light microscope (Leica DMI 4000B, Wetzlar, Germany).
Virus infection was validated after behavioral tests. Brain slices were mounted after incubation with a DNA-specific fluorescent probe (DAPI: 4′,6-diamidino-2-phenylindole, 1:1,000, Cell Signaling Technology). Fluorescence images were acquired using a laser scanning confocal microscope (model FV1000, Olympus Co., Ltd.)
9
Psoralen and Bakuchiol Modulate Osteoclastogenesis
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Psoralen and bakuchiol were obtained from the Food and Drug Verification Research Institute of China (Beijing, China). α-MEM was purchased from Hyclone (Hyclone Logan, UT, USA). FBS (Fetal Bovine Serum) was obtained from Biological Industries (Kibbutz Beit Haemek, Israel). M-CSF and RANKL were purchased from R&D Systems Inc. (Minneapolis, Minnesota, USA). TRACP and Cathepsin K Activity assay kits were obtained from Sigma (St. Louis, MO, USA). p-ERK, p-AKT, p-jun, p-p65, ERK, AKT, Jun, p65, rabbit IgG (Alexa Fluor 555-conjugate), GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody, DAPI (4, 6-diamidino-2-phenylindole), and ProLong® Gold Anti-Fade Reagent were purchased from Cell Signaling Technology (Danvers, MA, USA). PNPP-NA2 was purchased from Amresco (Cochran Road Solon, OH, USA). L(+)-tartaric acid was purchased from Solarbio (Beijing, China). SDS-PAGE (12%) was purchased from Invitrogen (Waltham, MA USA). Bone slices were purchased from Nordic Bioscience (Herlev, Denmark). ECL kit was purchased from GE Healthcare Life Sciences (Marlborough, USA). Psoralen and bakuchiol were weighed accurately and dissolved in DMSO to make a stock solution at a concentration of 0.05mM or 0.1mM (stored at -20°C). When the components were added to cells, the stock solution was diluted by MEM.
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