MEC cells were seeded in 6-well plates (5×104 cells) with DMEM/High glucose supplemented as previously described and treated with Cephaeline (IC50) in triplicates for 24 and 48h. After, cells were fixed with formaldehyde 4% for 15 min at room temperature. Blockage and cellular permeabilization were performed with 3% (w/v) bovine serum albumin (BSA) and 0.5% (v/v) Triton X-100 in PBS 1X for 1h. Anti- H3K9ac antibody (Cell Signaling Technology, Danvers, MA, USA) was diluted in (0.5% (v/v) Triton X-100 in PBS 1X and 1% (w/v) BSA) and incubated overnight. Subsequently, cells were washed and incubated with Alexa 488 secondary antibody (Cell Signaling Technology, Danvers, MA, USA) following with DNA staining using Hoechst 33342 (Cell Signaling Technology, Danvers, MA, USA). Ten fields of each slide were photographed and quantified. Images were taken using Nikon Eclipse Ti-S microscope and evaluated using Image J software (National Institute of Health, Bethesda, Maryland, USA).
Alexa 488 secondary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Alexa Fluor® 488 secondary antibody is a fluorescently labeled antibody that binds to the primary antibody, allowing for the visualization of target proteins or molecules in various applications such as immunofluorescence, flow cytometry, and western blotting. The Alexa Fluor® 488 dye has an excitation maximum at 495 nm and an emission maximum at 519 nm, providing green fluorescence that can be detected using standard fluorescein filter sets.
Automatically generated - may contain errors
Lab products found in correlation
Millicell ez slide, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Hoechst 33342, by Cell Signaling Technology (1 mentions)
Anti h3k9ac antibody, by Cell Signaling Technology (1 mentions)
Nf κb p65 antibodies igg, by Cell Signaling Technology (1 mentions)
Lps from escherichia coli 0111 b41, by Merck Group (1 mentions)
Fluoromount g mounting media, by Thermo Fisher Scientific (1 mentions)
Nf kb p65 primary antibody, by Cell Signaling Technology (1 mentions)
Paraformaldehyde pfa, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (1 mentions)
Triton x 100, by Santa Cruz Biotechnology (1 mentions)
Zen 2011, by Zeiss (1 mentions)
Alexa 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst, by ITW Reagents (1 mentions)
4 protocols using alexa 488 secondary antibody
1
Examining Histone H3K9ac Levels
2
Immunofluorescent Analysis of p65 Nuclear Translocation
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The immunofluorescent analysis of p65 nuclear translocation were performed in RAW 264.7 cells plated onto Millicell® EZ slides (Merck KGaA, Darmstadt, Germany) at 15,000 cells/cm2. Cells were exposed to 10% of PPP, BC, Alb-gel and RC for 1 hour following overnight serum starvation. Thereafter the cells were exposed to LPS from Escherichia coli 0111: B41 (Sigma Aldrich, St. Louis, MO) for another 1 hour. The cells were fixed with 4% paraformaldehyde, blocked with 1% bovine serum albumin (Sigma Aldrich, St. Louis, MO) and permeabilized with 0.3% TritonX-100 (Sigma Aldrich, St. Louis, MO). We used NF-κB p65 antibodies (IgG, 1:800, Cell Signaling Technology, #8242), at 4°C overnight. Detection was with the goat anti-rabbit Alexa 488 secondary antibody (1:1000, Cell Signaling Technology, #4412). Images were captured under a fluorescent microscope with a single filter block 455 nm (Oxion fluorescence, Euromex, Arnheim, Netherlands).
3
NF-kB Translocation Assay in HGF and HSC2 Cells
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Immunofluorescent analysis of nuclear factor-kappa B (NF-kB) p65 was performed in HGF and HSC2 cells plated onto Millicell® EZ slides (Merck KgaA, Darmstadt, Germany) at 1 × 104 cells/cm2. Cells were submitted to overnight serum starvation and then stimulated with IL1β + TNFα or the cell lysates for 1 h. Cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich, St. Louis, MO), blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO), and permeabilised with 0.3% TritonX-100 (Sigma-Aldrich, St. Louis, MO). Cells were subsequently incubated with NF-kB p65 primary antibody (1:400, Cell Signaling Technology, Cambridge, UK) at 4 °C overnight. Detection was carried out by incubation of Alexa 488 secondary antibody (1:800, Cell Signaling Technology, Cambridge, UK) for 1 h at room temperature. Fluoromount-G mounting media (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) containing DAPI was used to mount the glass slides. Images were captured under a fluorescent microscope with a dual excitation filter block DAPI-FITC (Echo Revolve Fluorescence microscope, San Diego, CA) to observe the nuclei translocation.
4
Confocal Microscopy of Stimulated Stem Cells
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Our group already described the general confocal microscopy procedure [24 (link)]. For this purpose, continuous and pulsatile electrically stimulated (Group 2a/b either for 3 or 7 days stimulation) as well as non-stimulated ASC, BMSC, and POB cells were fixed in 4% paraformaldehyde (Santa Cruz, Dallas, TX, USA), permeabilized with 0.1% Triton X-100 (Santa Cruz, Dallas, USA) and labeled with Phalloidin-Alexa 488 (Invitrogen) or focal adhesion kinase (FAK) primary antibody (#3285, Cell Signaling, USA) combined Alexa488 secondary antibody (Cell Signaling, Danvers, MA, USA) and counter-stained with Hoechst (PanReacAppliChem, Darmstadt, Germany). Microscopy was performed with an inverted confocal laser-scanning microscope (LSM780, Carl Zeiss, Jena, Germany). All images were taken at identical device settings to guarantee comparable results. The image processing was carried out using ZEN 2011 (Carl Zeiss Jena GmbH, Jena, Germany).
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