Axioimager m2 plus apotome2 microscope

BV-2 cells were seeded on glass cover slips and incubated with 10 μg/ml fluorescently labeled AMWAP or vehicle for different time spans prior to stimulation with 50 ng/ml LPS for 1 h. Cells were then fixed with 4% formaldehyde, washed with PBS, and incubated in blocking buffer containing 10% goat serum and 0.3% Triton X-100. Subsequently, cells were incubated with antibodies against Iba1 or the p65 subunit of NFκB in a solution containing 2.5% goat serum and 0.1% Triton X-100 for 1 h at room temperature. After 30-min incubation with goat anti-rabbit Alexa-594 (A-11012, Life Technologies), slides were washed with PBS and stained with 4′,6-diamidino-2-phenylindole (DAPI). Cover slips were mounted with fluorescent mounting medium (Dako Cytomation, Hamburg, Germany), and fluorescence photomicrographs were taken with an AxioImager.M2 plus ApoTome2 microscope (Carl Zeiss, Oberkochen, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to determine the ratio of nuclear to cytosolic NFκB p65 after quantifying pixel intensities of both cellular compartments and subtracting background fluorescence.

BV-2 microglial cells were seeded on cover slips in six-well plates and cultivated and stimulated as described before. Afterwards cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and F-actin was fluorescently labeled using 0.1 μg/ml Phalloidin-TRITC (Sigma-Aldrich). Nuclei were stained using DAPI, and the cover slips were mounted with fluorescent mounting medium (Dako Cytomation). Photomicrographs were taken with an AxioImager. M2 plus ApoTome2 microscope (Carl Zeiss). Quantitative scoring of microglial ramification was performed as described previously using a grid-cross analysis [6] (link).

BV-2 cells were seeded on sterile cover slips and were cultivated and stimulated as described before. Afterwards cells were fixed with 4% formaldehyde, rehydrated with PBS, and subsequently blocked with buffer, containing 10% goat serum and 0.3% Triton X-100 for 30 min. The cells were then incubated with primary antibody against p65 subunit of NF-κB diluted in PBS containing 2.5% goat serum and 0.1% Triton X-100 for 1 h at room temperature. Afterwards slides were washed with PBS and incubated with goat anti-rabbit Alexa-594 (A-11012, Life Technologies) for further 30 min. Nuclear DNA was stained with 4′, 6-diamidino-2-phenylindole (DAPI). Cover slips were mounted with fluorescent mounting medium (Dako Cytomation, Hamburg, Germany), and fluorescence photomicrographs were taken with an AxioImager. M2 plus ApoTome2 microscope (Carl Zeiss, Oberkochen, Germany). NF-κB translocation was determined measuring the mean fluorescence intensity of the nuclei and cytoplasmic areas using Image J. A nucleus/cytoplasma signal ratio above 1 indicates nuclear localization.