Phalloidin 594

Immunohistochemistry was performed either on alternate series of sections or in combination with ISH. Sections were washed in PBS, then in PGT (PBS with 0.2% gelatin and 0.25% Triton X-100) four times for 15 min. Sections were incubated overnight at 4°C with the following primary antibodies: anti-Tph2 (mouse monoclonal, 1/1000, Sigma-Aldrich), anti-5-HT (rabbit polyclonal, 1/1000, Sigma-Aldrich), anti–serotonin transporter (anti-SERT; rabbit polyclonal, 1/1000, Calbiochem). For fluorescence microscopy, sections were then incubated for 2 h at room temperature with the following secondary antibodies: donkey anti-rabbit 488 (1/200, The Jackson Laboratory), donkey anti-rabbit Cy3 (1/200, The Jackson Laboratory), donkey anti-mouse 488 (1/200, The Jackson Laboratory), donkey anti-mouse Cy3 (1/200, The Jackson Laboratory), or phalloidin 594 (1/40, Invitrogen). Sections were rinsed in PB, mounted in mowiol-Dabco (25 mg/ml), and stored at 4°C.

Cells were seeded on 18 mm glass coverslips, fixed with 4% methanol-free paraformaldehyde, and blocked in 0.1% BSA + 0.01% TritonX-100 for 1 hr. Cells were probed with anti-CLCA1 (Abcam, ab180851, 1:100), anti-Muc2 (Santa Cruz, sc-515032, 1:200), anti-Rab7 (Sigma, R4779, 1:400), and Phalloidin-594 (Invitrogen, A12381, 1:400) at 4℃ overnight. Cells were further incubated with fluorophore tagged secondary antibodies for 2 hr. Nucleic acid was stained with DAPI (1 µg/ml). Coverslips were mounted with Prolong Gold Antifade Reagent (Thermo Fisher) and visualized in confocal microscope (Leica SP8) for immunocytochemistry and in Elyra PS1 (Carl Zeiss) for structured illumination microscopy.

BMECs (400 μL) were seeded at 5.0 × 10⁵ cells/mL on 35 mm glass bottom dish coated with 0.1% polylysine, and cultured for 2 days to reach confluence. The treatment plan was described in Section 2.4. After treatment, cells were first washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After the removal of excessive paraformaldehyde, the fixed cells were incubated in a fresh blocking buffer (0.5% Triton X-100 in PBS, pH 7.4, containing 10% normal goat serum) for 1 h at room temperature. The cells were incubated overnight at 4 °C with anti-claudin-5 (Thermo Fisher Catalog # 34-1600, Waltham, MA, USA) or anti-ZO-1 (Proteintech Catalog (Rosemont, IL, USA): 20742-1-AP) primary antibody, which were diluted 1:50 (anti-claudin-5) or 1:20 (ZO-1) in PBS with 3% bovine serum albumin. The primary antibodies were detected by incubation with Alexa Fluor 488 (1:300) in PBS containing 3% bovine serum albumin. Cells were then incubated with phalloidin-594 (Invitrogen) for 30 min at room temperature in the dark. After that, the cell nucleus was stained with 4, 6-diamidino-2-phenylindole (DAPI). Images were captured on an Olympus FV1200 laser scanning confocal microscopy (Olympus, Tokyo, Japan).

To assess the migration of GRC cells cultured in microfluidic chips, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.1% Triton X-100 for 20 min. Actin filaments and nuclei were stained with phalloidin-594 (1:40; Invitrogen, United States) and Hoechst 33342 (1:1500; Thermo Fisher Scientific, United States), respectively. Fluorescent cell images were obtained using a high-content screening microscope (Celena X; Logos Biosystems, Republic of Korea). A set of images acquired from the chips, representing cell mobility and the surrounding tissue, was taken at 16 repetitive regions of interest (ROI) between the trapezium-shaped pillars in the chips. Among the images, grayscale fluorescence images of F-actin staining (red channel) were used for subsequent image analyses. In addition, each ROI was image-captured along the z-axis using 27 slices within full thickness. Six microfluidic chips were used for each GRC level, and 2,592 raw images were captured from a single microfluidic chip.

Primary antibodies: mouse anti-EEA1 ( ICC 1:300, novus biologicals 6D4.1D4), mouse anti-LAMP2 (1:200 BD biosciences 555803), rabbit anti-GFP (IP 1:100 abcam AB290), mouse anti-VPS35 (ICC 1:200, IP 1:50 Santa Cruz, sc-374372), rabbit anti-RET finger protein/TRIM27 (ICC 1:200, IP 1:50, IBL18791), rabbit anti-Parkin (WB 1:1000, novus biologicals NBP2-41287), rabbit anti-HA-Tag (ICC 1:800, cell signaling technologies, C29F4).
Secondary antibodies used for ICC: green goat anti-rabbit IgG (H + L)488 (1:200, Thermo Fisher Scientific, A11088) and Cy3 AffiniPure donkey anti-mouse IgG (H + L) (1:200, Jackson Laboratories, 715–165-150), donkey anti-rabbit cy3 (H + L) (1:200, Jackson Laboratories 711–165-151), phalloidin 594 1:40 (Invitrogen A12381).
Secondary antibodies used for WB (1:10 000): red IRDye 680RD goat anti-rabbit IgG (H + L) (LI-COR Biosciences, 926–68,071) and green IRDye 800CW goat anti-mouse IgG (H + L) (LI-COR Biosciences, 926–32210).