Goat anti mouse cy3

Cells were fixed with 4% paraformaldehyde (10 min at room temperature) followed by permeabilization with 0.1% Triton X-100 for 10 min. Cells were washed three times with PBS/3%BSA and blocked for 20 min with the same solution. The endoplasmic reticulum was visualized using mouse anti-calnexin AF18 (Abcam) and goat anti-mouse Cy3 labeled antibody (Dianova), the Golgi apparatus was visualized using rabbit anti-α-Mannosidase II [83 (link)] and goat anti-rabbit Cy3 labeled antibody (Dianova). The IBs were detected with mouse anti-c-myc 9E10 (Santa Cruz Biotechnology) and goat anti-mouse Cy3 labeled antibody (Dianova) or directly with goat anti-c-myc FITC conjugated antibody (Novus Biologicals). Flag-HA-tagged polySTs were detected with mouse anti-FLAG M5 (Santa Cruz Biotechnology) and goat anti-mouse Cy3 or goat anti-mouse FITC labeled antibody. Primary antibodies were incubated for 2 h and secondary antibodies for 1 h (diluted in 3% BSA/PBS) at room temperature. In between, cells were washed 5 times with PBS-0.05%Tween. For analysis by laser scanning confocal microscopy, cells were coated with Vectashield Mounting Medium (Vector Laboratories).

Cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. Staining was performed using antibodies against: ApoB (Santa Cruz, Heidelberg, Germany), VE-Cadherin (BD Biosciences, Heidelberg, Germany), MRP2 (Cell Signaling, Leiden, Netherlands), and ZO-1 (Life Technologies, Darmstadt, Germany) and secondary antibodies goat-anti-mouse-Cy3 (Santa Cruz, Heidelberg, Germany), goat-anti-rabbit-AlexaFluor488 (Life Technologies, Darmstadt, Germany), AlexaFluor633 phalloidin (Life Technologies, Darmstadt, Germany), and DAPI (Life Technologies, Darmstadt, Germany). Samples were enclosed into fluorescent mounting medium (Dako, Hamburg, Germany). Cells were analyzed using an AXIO Observer Z1 fluorescence microscope with Apotome 2 extension (Carl Zeiss AG, Jena, Germany). The software FiJi (GNU General Public License) was used for image analysis.