Ma1 118

For cell body visualization, 21 DIV neurons were fixed with a 4% paraformaldehyde (PFA) (Sigma–Aldrich) solution in PBS (Sigma–Aldrich) for 20 min. The cells were rinsed five times with PBS and subsequently permeabilized and blocked with a 3% bovine serum albumin (BSA) (Sigma–Aldrich) and 0.1% Triton X-100 (Sigma–Aldrich) solution in PBS for 45 min. Neuronal bodies and projections were labeled with antibodies against beta-3 tubulin (mouse monoclonal, Thermo Fisher, MA1-118, dilution 1:200; secondary antibody: goat anti-mouse IgG (H + L) Alexa Fluor 488, Life Technologies, dilution 1:1000), MAP2 (rabbit polyclonal, Thermo Fisher, PA5-17646, dilution 1:1000; secondary antibody: goat anti-rabbit Alexa Fluor 488, Life Technologies, dilution 1:1000) and Tau (mouse monoclonal, Thermo Fisher, AHB0042, dilution 1:1000; secondary antibody: goat anti-mouse Alexa Fluor 647, Life Technologies, dilution 1:1000). Image acquisition was performed with an AxiObserver A1 Microscope (Zeiss, Germany) using 10x and 20x objectives.

To evaluate the integrity of the primary hippocampal neurons inside our microfluidic chips, some chips were fixed in PBS containing 4% paraformaldehyde (PFA, Electron Microscopy Science 15714S) and 4% sucrose (Sigma S9378) for 8 min at room temperature and rinsed twice in PBS for 5 min. Then, they were permeabilized for 30 min at room temperature with 0.2% Triton (Sigma X100) and 1% bovine serum albumin (BSA, Thermofisher 15561020) in PBS. After removing the permeabilizing solution, primary antibodies were added in PBS with 1% BSA and the samples incubated at 4 °C overnight. The samples were rinsed for 5 min with 1% BSA in PBS and further incubated with the corresponding secondary antibodies for 2 h in PBS with 1% BSA at room temperature. Finally, the chips were rinsed first with 1% BSA in PBS and last only with PBS. The following primary antibodies and dye were used: DAPI (4’,6-diamidino-2-phénylindole, nuclear DNA staining) (1050, Euromedex, 1/2000), MAP2 (Microtubule-associated protein 2) (M4403, Sigma, 1/1000), β3-tubulin (MA1-118, Thermofisher, 1/1000) and GFAP (Glial Fibrillary Acidic Protein) (Z0334, Dako, 1/1000). Species-specific secondary antibodies coupled to Alexa 350, 488, 555 or 633 were used (1/1000, Invitrogen) to visualize bound primary antibodies.

Cells were fixed in 4% PFA and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). The antibodies against muscle actin (M0635, Agilent, Santa Clara, CA, USA), beta-3 tubulin (MA1-118, Thermo Fisher Scientific), CDX2 (12306, Cell Signaling Technology, Danvers, MA, USA), goat anti-mouse IgG, Alexa Fluor 568 (H + L) (A-11019, Thermo Fisher Scientific), goat anti-rabbit IgG, Alexa Fluor 568 (H + L) (A-11036, Thermo Fisher Scientific) were used. The nuclei were stained with Hoechst 33,258 (Thermo Fisher Scientific). Images of the stained cells were captured by using a confocal microscope (LSM 700, Carl Zeiss AG, Oberkochen, Germany) at the Centre for PanorOmic Sciences, the University of Hong Kong.