A11011

Mature microvascular networks were rinsed with warm PBS followed by the addition of approximately 100 μl of 4% paraformaldehyde (Electron Microscopy Sciences, # 15700) to the media channels and left at room temperature. After 15 min of fixation, devices were rinsed twice with PBS, and blocking solution (4% bovine serum albumin, 0.5% goat serum) (Sigma-Aldrich) was added. Devices were incubated for 1 day at 4°C, washed with PBS, and stained with primary antibodies: ICAM-1 (Biolegend, 4453320),VCAM-1 (Abcam, ab134047), CD31 (Abcam, ab28364), conjugated Alexa Fluor 647 anti-human CD326 (EPCAM) (BioLegend, 324212), Acti-stain 555 phalloidin, F-actin (Cytoskeleton, PHDH1-A) and incubated at 4°C for another day. Devices were again washed with PBS and secondary antibodies (Thermo Fisher Scientific, A-11070, A-11011, A-21052) DAPI (4′,6-Diamidino-2-Phenylindole, Dihydrochloride, Invitrogen) or DyLight 649 labeled Ulex Europaeus Agglutinin I (Vector Laboratories) were added, followed by incubation at 4°C protected from light. Finally, samples were washed again with PBS and 3D images were acquired with a confocal microscope (Olympus FV1000) at 20×. Z-stacks were collapsed with maximum intensity projections for viewing (800 × 800 pixels) using FIJI (22 (link)).

Chick eggs were incubated for 25-29 h at 38°C until embryos reached the desired stages. Eggs were windowed, a small amount of Pelikan Fount India ink (diluted 1:5 with Tyrode's solution) was injected beneath the embryo into the yolk sac to increase the embryo's visibility, the vitelline membrane was locally removed, and the plasmids pEx-FGF8 and pEx-dnFGFR, respectively (a kind gift from E. Grove, The University of Chicago, IL, USA), were injected together with pCAβ-eGFP and Fast Green (1 mg/ml for each plasmid) into the anterior neural tube or between the anterior neural folds of the embryo. Two platinum-iridium electrodes were placed on either side of the embryo and four 10 V pulses of 20 ms were supplied to transfect the right side of the neural folds/tube. Eggs were re-sealed and incubated for another 24-48 h before dissection and fixation. Only embryos that had a distinct recognisable diencephalic vesicle and did not show gross overall brain abnormalities were selected for analysis. Post in situ hybridisation staining for GFP was performed using rabbit anti-GFP antiserum at a concentration of 1:200 (Invitrogen, ThermoFisher; A-6455) followed by incubation with a 1:200 dilution of secondary antibody coupled to Alexa Fluor 488/568 (Invitrogen; A32731 and A-11011)

To visualize cilial structures, immunofluorescence was performed using an anti-acetylatedα-tubulin antibody (1:1000, T6793, Sigma) and an anti-γ-tubulin antibody (1:1000, T3320, Sigma) [67 (link)]. Briefly, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature. Fixed cells were permeabilized with 0.05% Triton X-100 and then incubated with the primary antibodies overnight at 4° C. Alexa Fluor 568-conjugated anti-rabbit (1:1000, A-11011, Invitrogen) or Alexa Fluor 647-conjugated anti-mouse (1:1000, A-21235, Invitrogen) antibodies were used as secondary antibody. Counter staining of nuclei was done with DAPI (Sigma) [68 (link)]. The experiments were conducted in quadruplicate. The same treatment was used for Ki67 (1:1000, J3009, Santa Cruz Biotechnology, USA), PCNA (1:1000, A5324, Selleckchem, USA), β-catenin (1:200, A5038, Bimake, USA) staining and Wnt3a (1:200, 2721, Cell Signaling Technology, USA) staining as well as for Osteocalcin (OCN) (1:1000, 16157-1-AP, Proteintech, USA) staining. Ki67 was visualized using a FITC-conjugated goat anti-rabbit (1:1000, 0110119-0100, BBI) antibody.

Embryos (n = 38, 3 replicates) were fixed in 3.7% paraformaldehyde for 20 min at room temperature, permeabilized with PBS/PVA containing 0.5% Triton X-100 at 37 °C for 1 h, and then incubated in PBS/PVA containing 1.0% bovine serum albumin at 37 °C for 1 h. Subsequently, the embryos were incubated overnight at 4 °C with anti-LC3 (ab58610, 1:100; Abcam, Cambridge, UK), anti-cytochrome C (ab110325, 1:100; Abcam), anti-p53 (sc6243, 1:100; Santa Cruz Biotech, CA, USA) and anti-OCT4 (sc8628, 1:100; Santa Cruz Biotech) antibodies. To check the fluorescence signal of 5mC, blastocysts were denatured with 1 N HCl at room temperature for 30 min and neutralized with 0.1 M Tris-HCl, pH 8.0 for 15 min. Subsequently, blastocysts were incubated in PBS containing 1% BSA, and then incubated overnight at 4 °C with 5mC antibody (ab10805, 1:100, Abcam, Cambridge, UK). After washing three times with PBS/PVA, the oocytes and embryos were incubated at 37 °C for 1 h with either goat anti-rabbit IgG (A11011, 1:200, Invitrogen) or rabbit anti-goat IgG (A11079, 1:200, Invitrogen), anti-mouse IgG (A21202, 1:200, Invitrogen). The oocytes and embryos were then stained with Hoechst 33342 for 5 min, washed three times with PBS/PVA, mounted onto slides, and examined using a confocal microscope (Zeiss LSM 710 META, Jena, Germany). Images were processed using Zen software (version 8.0, Zeiss, Jena, Germany).