Immu mount

TM cells were plated on glass coverslips in a 6-well plate and when they reached 100% confluence the medium was switched to DMEM with 1% FBS for 4 days. Cells were then treated with BIM (1000 µM) or vehicle for 24 hours prior to fixing with 4% paraformaldehyde for 10 minutes. Following 3 washes with phosphate-buffered saline (PBS), cells on coverslips were blocked in Cell Signaling Blocking Buffer (PBS with 5% goat serum and 0.3% Triton X-100) for 1 hour. Cells on coverslips were then incubated overnight at 4°C with anti-fibronectin IgGs conjugated to Alexa Fluor 488 (Cell Signaling) in Cell Signaling Antibody dilution buffer (PBS with 1% BSA and 0.3% Triton X-100). Following three washes with PBS, cells on coverslips were DAPI stained then mounted onto slides with Immu-Mount (Epredia, Portsmouth, NH, USA) and sealed with nail polish. Treated and control cells were imaged using confocal microscopy during the same session, at identical settings.

For immunofluorescence staining of serotonin after sortilin inhibition, 2,000 BON cells were trypsinized, seeded onto glass slides, and fixed with methanol/acetone (1:1) for 2 min at room temperature. After washing with PBS and blocking with 5% goat serum in PBS (Biochrom) for 30 min at room temperature, the cells were incubated with the primary antibody against serotonin (1:400, #M0758, clone 5HT-H209, Dako) for 1 h at room temperature. The cells were washed three times for 2 min with PBS and incubated with Cy3-Goat anti-mouse IgG (1:1000, Jackson Immuno Research, 111-225-144) for 30 min at room temperature. After repeated washing with PBS, the cells were incubated with ethanol for 2 min and mounted in Immu-Mount™ (#9990402, Epredia).
All immunofluorescence images were acquired with the confocal laser scanning microscope TCS SP8 (Leica) or Observer 7 microscope (Zeiss). Images were collected and analyzed with Leica Application Suite X 3.5.6.21594 (LAS X, Leica) and ZEN 3.4 (Zeiss). Five images were collected per sample.

To visualize biotinylated (EIS)₂-RGD6 in animals used to perform experiment (EIS)₂-RGD6 II, a set of parallel sections per animal were first processed for the visualization of GFAP following the aforementioned fluorescence-based simple immunohistochemistry protocol. The following primary and secondary antibodies were used: monoclonal mouse anti-GFAP (G3893, Sigma-Aldrich, Steinheim, Germany; 1:500) and Dylight488-linked goat anti-mouse (ab96879, Abcam, Cambridge, UK). Subsequently, sections were immersed in buffer blocking—10% foetal bovine serum (10500064, Fisher Scientific, Madrid, Spain), 0.3% bovine serum albumin (A7906, Sigma Aldrich, Steinheim, Germany), 0.3% Triton X-100 (X100, Sigma Aldrich, Steinheim, Germany) in TBS—for 1 h at room temperature (RT), incubated with alexa594-linked streptavidin (S11227, Thermo Scientific, Paisley, UK; 1:500) for 1 h at RT and, after several washes, with DAPI (62247, Thermo Scientific, Paisley, UK; 1:5000) for 5 min at RT. Finally, sections were cover-slipped with Immumount (9990402, Epredia, Breda, The Netherlands).