Goat anti mouse cy3

The IRF-3 translocation assay was performed as described elsewhere [65 (link)]. Briefly, MA-104 cells were transfected with plasmids encoding 250 ng GFP-IRF-3 and either EV or 1000 ng of FLAG-tagged PLP-expressing plasmids using Fugene HD (Promega). To activate the IFN pathway, cells were infected with RVFV Cl 13 and an MOI of 5 at 17 hpt. At 8 hpi cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. Immunofluorescence analysis was done as in [68 ]. Samples transfected with EV were treated with anti-RVFV mouse serum (Friedemann Weber, University of Gießen, [53 (link)]) and goat anti-mouse Cy3 (Dianova GmbH) secondary antibody. PLP-expressing cells were stained with mouse anti-FLAG (Sigma-Aldrich) and goat anti-mouse Cy3 (Dianova GmbH) antibodies. Samples were analyzed by a fluorescence microscope (Zeiss). Depending on the number of transfected cells, at least three images were taken, and the number of cells double-positive for GFP and FLAG was divided by the number of cells showing IRF-3 nuclear translocations.

For staining of cells of the hepatic cell layer the sealing foil of the biochip (serving as cell substrate) was carefully removed with a scalpel. For staining of cells from the vascular layer the suspended membrane was similarly removed. Cells were then fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Staining was done with antibodies against: MRP-2, PECAM-1 (Cell Signaling, Leiden, The Netherlands), von Willebrand factor, VE-Cadherin (BD Biosciences), ApoB (Santa Cruz, Heidelberg, Germany), ZO-1 (Life Technologies, Karlsruhe, Germany), CYP3A4 (Merck-Millipore, Schwalbach, Germany) CD163 (Biolegend, United Kingdom), CD197 (BD BioScience), CD68 (Santa Cruz Biotechnology, Heidelberg, Germany) and secondary antibodies goat-anti-mouse-Cy3, goat-anti-rabbit-Cy5 (Dianova, Hamburg, Germany), goat-anti-rabbit-AlexaFluor488 and DAPI (Life Technologies). Samples were embedded into fluorescent mounting medium (Dako, Hamburg, Germany). MRP-2 activity analysis was performed by incubation of HepaRG cell layers in serum free Williams E medium (GIBCO) containing 5 μM 5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate (CD-FDA) (Sigma-Aldrich) at 37 °C for 15 min. Subsequently, imaging was performed on an AXIO Observer Z1 fluorescence microscope with Apotome 2 extension (Carl Zeiss AG, Jena, Germany). Image analysis was done with ImageJ2 software.

Cells and cultured neurons on glass coverslips were fixed with 4% PFA/4% sucrose in PBS pH 7.4 for 20 min at RT. Afterwards, the cells were blocked and permeabilized with 5% NGS/0.2% Triton X-100 in PBS for 30 min. The primary antibodies against GFP (SC8224, RRID:AB_2276004, 1:500, Santa Cruz), gephyrin (147011, RRID:AB_887719, 1:500, Synaptic Systems), GlyRα1 (146111 or 146118, RRID:AB_887723 or RRID:AB_2832240, 1:500, Synaptic Systems), pan-α-GlyR (146011, RRID:AB_887721, 1:250, Synaptic Systems), and anti-mEos-488 (N3102-At488, 1:200, NanoTag) in blocking solution were incubated for 1 h, followed by incubation with the secondary antibodies goat-anti-rabbit-Alexa-488 (111-546-003, RRID:AB_2338053, Dianova), goat-anti-mouse-Cy3 (115-165-003, RRID:AB_2338680, Dianova), goat-anti-rabbit-Cy3 (111-165-003, RRID:AB_2338000, Dianova), goat-anti-mouse-Cy5 (115-175-146, Dianova), or goat-anti-rabbit-Cy5 (111-175-006, Dianova) diluted 1:500 in a blocking solution for 1 h in the dark. The cell nuclei were stained with DAPI in PBS for 5 min, and the cells were mounted on glass slides in Mowiol.