Imagexpress micro xls automated microscope

Cells in 96-well plates were fixed in 4% paraformaldehyde for 15 min and washed with PBS with Triton™ X-100 (0.1%; Sigma, MO, USA) (PBS-T). Primary antibodies, diluted appropriately in immunobuffer (PBS with 0.2% Triton™ X-100, 1% goat serum (Gibco, CA, USA) and 0.04% thimerosal (Sigma, MO, USA)), were added overnight at 4 °C (See Additional file 2: Table S3 for details). The following day, secondary antibodies were added at room temperature for 3 h. Hoechst nuclear counterstain (1 μM; Hoechst 33258, Sigma, MO, USA) in Tris-NaCl-EDTA (TNE) buffer was added for 10 min at room temperature. For stains that were sensitive to detergent (zona occludens-1; ZO-1), Triton™ X-100 was excluded until after the primary antibody incubation. Wells were then washed again and counterstained with Hoechst-33258 nuclear stain. For experiments directly comparing endothelial and pericyte inflammatory response, both endothelia and pericytes were seeded and imaged on the same plate. Images were acquired on an ImageXpress Micro XLS™ automated microscope (Molecular Devices).

Cells in 96-well plates were fixed in 4% paraformaldehyde (PFA) for 15 min, and when endosomal markers (EEA1, Rab5, Rab7, LAMP1) were stained, defatted with ice-cold methanol for a further 10 min at 4 °C. Cells were washed with PBS-Triton™ X-100 (0.1%; Sigma) (PBS-T). Primary antibodies, diluted appropriately in immunobuffer (PBS with 0.2% Triton™ X-100, 1% goat serum (Gibco), and 0.04% thimerosal (Sigma)), were added overnight at 4 °C (See Supplementary Table 4 for details). The following day, species-specific secondary antibodies and Hoechst nuclear counterstain (1 µM; Hoechst 33258, Sigma) in immunobuffer were added at room temperature for three hours. Wells were then washed again, and images were acquired on an ImageXpress Micro XLS™ automated microscope (Molecular Devices).