Dylight 549 anti mouse

Immunocytochemistry of cultured human astrocytes was performed as previously reported [16 (link), 18 (link)]. Briefly, cells were seeded and grown in sterile glass coverslips on 24-well plates and treated as described above (4 h incubation with Psy 20 µM or Psy 20 µM + LCN 1 nM; untreated astrocytes were used as controls). After treatment, cells were washed and fixed in 4% PFA for 5 min on ice. Cells were then washed in PBS and permeabilized with 0.1% Triton-X100 in PBS for 5 min, room temperature (RT). Afterwards, cells were blocked with 1% BSA in PBS + 0.1% Triton-X100, at 4 °C overnight. Following blocking, samples were incubated with primary antibody mouse anti-vimentin (sc373717, Santa Cruz Biotechnology; dilution 1:800) and with secondary antibody Dylight 549 anti-mouse (#715–505-020, Jackson ImmunoResearch, Ely, UK; dilution 1:1000). Cells were when washed and incubated with DAPI (#62,248, Thermo Fisher Scientific; dilution 1:500) for nuclear staining and mounted on a microscope slide using antifade reagent (S36936, Thermo Fisher Scientific). Coverslips were sealed and samples were stored at 4 °C until imaged using an Olympus Bx51 upright fluorescence microscope at 20× magnitude. Image acquisition settings were kept constant across all treatments. Image analysis of fluorescence was made using the ImageJ software version 1.52a (https://imagej.nih.gov/ij).