Af3369

For immunofluorescence analyses, vascularized organoids were fixed in 4% PFA solution for 30 min, washed in PBS and embedded into an agarose gel (1%). Afterward, 5 µm paraffin sections were prepared. Sections were deparaffinized, rehydrated and stained with eosin and hematoxylin. For immunofluorescence analyses, antigens were unmasked using Sodium Citrate buffer (10 mM, pH6). Primary antibodies to TUJ1 (Biozol, 801202), GFAP (DAKO, Z0334), CD31 (DAKO, M0823), Iba1 (WAKO, 019-19741), Sox1 (R&D Systems, AF3369), Pax6 (Biolegend, 901301), Brachyury (T) (R&D Systems, AF2085), MAP2 (Abcam, AB32454), N-Cadherin (Sigma-Aldrich, C3865) and NG2 (Merck-Millipore, AB5320) were used. Sections were incubated with primary antibodies overnight at 4 °C. Secondary Cy2‐, Cy3- or Cy5-labelled antibodies were used to visualize primary antibodies. Sections were incubated with secondary antibodies for 2 h at room temperature. All antibodies were diluted in blocking solution.

Cells were fixed with 4% formaldehyde for 15 min and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were blocked with 10% donkey serum in PBS for at least 1 h. Primary antibodies against Nestin (Abcam, ab92391), SOX2 (Abcam, ab59776), SOX1 (R&D Systems, AF3369), PAX6 (BioLegend, PRB-278P), Ki67 (CST, 9449S), Dlx2 (Abcam, ab117546), FOXG1 (Abcam, ab18259), MAP2 (Sigma Aldrich M9942), TBR1 (Abcam ab31940), BRN2 (Cell Signaling 12,137), NeuN (Abcam ab104225), SOX9 (R&D Systems AF3075), GLT1 (Novus Biologicals NBP1–20136), and GFAP (Dako Z0334) were diluted in blocking buffer and incubated overnight at 4 °C. Following several washes, the cells were incubated with the appropriate donkey anti-rabbit IgG, anti-mouse IgG, or anti-goat conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 secondary antibodies (Life Technologies) for 1 h at room temperature. Cells were counterstained with DAPI and visualized with the Leica DM6000 inverted microscope. Images were acquired using the Q-Imaging Retiga Xi Firewire High-Speed, 12-bit cooled CCD camera and Volocity software. Staining was quantified using ImageJ software and represented as a portion of total nuclei. On average, 2.5 × 10³ cells were quantified from each experiment. Results are shown with the standard deviation of the mean of three independent experiments.