Goat anti rabbit cy5

Immunofluorescent DSB and repair protein staining has been described in detail elsewhere [36 (link),76 (link)]. In brief: the cells fixed in ethanol were subjected to cyto-centrifugation followed by immunofluorescent staining to detect DNA damage-associated protein accumulation as microscopic foci at DNA double-strand break sites as previously described [77 (link)]. Primary antibodies against γ-H2AX (Mouse anti-γ-H2AX; Merck Chemicals), rabbit anti-MRE11 (Novus), rabbit anti-phospho-ATM (pS1981; abcam), and 53BP1 (Rabbit anti-53BP1; Novus) were applied and detected with secondary goat anti-mouse Alexa-488 (Mobitec, Göttingen, Germany), goat-anti-rabbit-Cy5 or goat-anti-mouse Cy3-labeled antibodies (both Dianova). For SMLM, preparations were embedded in ProLong Gold anti-fade solution (Thermo Fisher Scientific, Schwerte, Germany). Wide field fluorescence images were recorded using a Zeiss Axioimager 2i fluorescence microscope system (ISIS; MetaSystems, Altlussheim, Germany). Cells that showed deformed or overlapping nuclei were excluded from analysis.

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.

Seven μm thick paraffin sections of human MS brain (n = 12) and control brain tissue (n = 12) were obtained from the Multiple Sclerosis and Parkinson’s Tissue Bank, Centre for Brain Sciences, Imperial College London. Paraffin-embedded tissue from normal human prostate was obtained from the Department of Pathology, University Hospital of Hamburg-Eppendorf, Hamburg. The sections were dehydrated in a descending alcohol series prior to IHC. Epitope retrieval was performed in 0.1 M citrate buffer. Sections were blocked with 5% NGS (Vector Laboratories) in PBS at room temperature for 2 h. Sections were then incubated with the primary antibodies directed against CEACAM1 (clone C5-1X; Reliatech; diluted 1:50) and CD20 (Fisher Scientific; diluted 1:400) in PBS at 4 °C overnight. As a control, sections were incubated in the absence of primary antibody. The next day, sections were stained with goat anti-rabbit Cy5 (Dianova; diluted 1:300) and goat anti-mouse Cy3 (Dianova; diluted 1:600) at room temperature for 2 h. Counterstaining of cellular nuclei was performed by incubation with 4′,6-diamidino-2-phenylindole (DAPI, Roche; diluted 1:5,000 in PBS). Sections were analyzed on a Keyence Biorevo BZ-9000 microscope.