Astrocytes were seeded onto 12-mm glass coverslips placed in 24-well plates containing growth medium; then, this medium was removed, and the cells were allowed to incubate in serum-free medium overnight. The astrocytes were then incubated with 0.1% DMSO (vehicle) or with statins (5 µg/mL) for 1 hour followed by incubation with TGF-β2 (5 ng/mL) for 1 hour. The cells were then fixed using 4% paraformaldehyde and permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich). Cells were incubated overnight at 4°C with a pMLC (S19) antibody (Cell Signaling Technology) in PBS containing 2% BSA. After several washes with PBS, the cells were incubated with cy-3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) in PBS containing 2% BSA for 1 hour at room temperature. Cell nuclei were counterstained with mounting media containing 1 µg/mL of 4′,6-diamidino-2-phenylinodole (DAPI) (Thermo Fisher Scientific). Images were acquired using an LSM710 confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) and processed using Zeiss ZEN 2012 imaging software.
4 6 diamidino 2 phenylinodole dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States
4′,6-diamidino-2-phenylinodole (DAPI) is a fluorescent stain used in molecular biology to label and visualize DNA. It binds strongly to adenine-thymine (A-T) rich regions of DNA, emitting a blue fluorescence when excited by ultraviolet (UV) light.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Cy3 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ripa lysis buffer, by Elpis Biotech (1 mentions)
Anti gapdh, by AbFrontier (1 mentions)
Rabbit anti neun monoclonal antibody, by Merck Group (1 mentions)
Anti gfap, by Agilent Technologies (1 mentions)
2 protocols using 4 6 diamidino 2 phenylinodole dapi
1
Statin Effects on Astrocyte Cytoskeleton
2
Protein Isolation and Immunodetection Protocol
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Protein isolation with RIPA lysis buffer was purchased from Elpis biotech (#EBA-1149, Deajeon, Korea) and Pierce™ BCA Protein Assay Kit was purchased from Thermo (#23227, MA, USA). Anti-GFAP, rabbit polyclonal antibody was purchased from DAKO (#Z0334, CA, USA). Anti-NeuN rabbit monoclonal antibody was purchased from Millipore (#3838, MA, USA). Anti-PSD-95, mouse monoclonal antibody, anti-rabbit, goat polyclonal tagged Alexa Fluor 488, anti-mouse, goat polyclonal tagged Alexa Fluor 555 and 4'. 6-diamidino-2phenylinodole (DAPI) were purchased from ThermoFisher (#MA1-046, #A11034, #A11012, #A21426 and #D3571, MA, USA). Anti-GAPDH, rabbit polyclonal antibody was purchased from AbFrontier (#LF-PA0018, Seoul, Korea). Rabbit polyclonal antibody was purchased from WAKO (#016-20001, Osaka, Japan). Antimouse, sheep polyclonal horseradish peroxidase (HRP) tagged antibody was purchased from Abcam (#ab26116, #ab26113 and #ab6808, EA, UK). Anti-p-STAT3 (Tyr 705), STAT3, p-p44/42, p44/42 rabbit polyclonal antibody was purchased from Cell signaling (#9145, #12640, #4377 and #4695 MA, USA). Anti-NF-κB mouse monoclonal were purchased from Santa Cruz (#sc-7151, Taxas, USA)
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