Anti nestin

Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F12), Neurobasal Medium, B-27 supplement, phosphate-buffered saline (PBS) buffer, fetal bovine serum (FBS) and anti-glial fibrillary acidic protein (GFAP) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Recombinant mouse epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were purchased from R&D Systems (Minneapolis, MN, USA). Poly-L-ornithine (PLO), laminin and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-Nestin and oligodendrocyte marker O4 (O4) antibodies were purchased from Millipore (Burlington, MA, USA). Anti-Sox2 and Ki67 antibodies were purchased from Abcam (Cambridge, UK). Anti-ionized calcium-binding adaptor molecule 1 (Iba1) was purchased from FUJIFILM Wako Chemicals (Richmond, VA, USA). Growth-factor-reduced Matrigel was purchased from BD Biosciences (Bedford, MA, USA). Alexa 568 goat anti-mouse, Alexa 568 goat anti-rat, Alexa 488 goat anti-rabbit, and Alexa 488 and 568 goat anti-rabbit secondary antibodies were purchased from Life Technologies (Carlsbad, CA, USA). WKYMVm (Trp-Lys-Tyr-Met-Val-D-Met-NH₂) was purchased from Anygen Inc. (Gwangju, Korea).

To evaluate the expression of stemness and differentiation markers, the following antibodies were used: anti-CD133 (1:50, Santa Cruz Biotechnology, Dallas, TX, USA); anti-nestin (1:50, Millipore, Burlington, MA, USA,); anti-GFAP (1:200, DakoCytomation, Glostrup, Denmark); anti-βIII Tubulin (1:100, Cell Signaling, Danvers, MA, USA); anti-MBP (1:50, Santa Cruz Biotechnology, Dallas, TX, USA). Each marker was analyzed in a separate set of experiments and with at least two replicates.

The following antibodies were used: anti-ALB (Abcam, ab8940, 1:400); anti-β-actin (Santa Cruz Biotechnology, sc-130301, 1:5 000); anti-caldesmon (Sigma-Aldrich, C4562, 1:300); anti-CD73 (BD Biosciences, 550741, 1:50); anti-CD90 (BD Biosciences, 555595, 1:100); anti-CD105 (eBioscience, 1-1057, 1:100); anti-CENPA (Abcam, ab13939, 1:400); anti-fibrillarin (Abcam, ab4566, 1:100); anti-FLAG (Sigma-Aldrich, M2, 1:2 000 for western blotting, 1:400 for immunofluorescence); anti-GAL4 (Abcam, ab14477, 1:1 000 for western blotting, 1:400 for immunofluorescence); anti-γH2AX (Millipore, 05-636, 1:400); anti-IgG-APC (eBioscience, 555751, 1:100); anti-IgG-FITC (eBioscience, 555748, 1:100); anti-IgG-PE (eBioscience, 555749, 1:100); anti-MAP2 (Sigma-Aldrich, m4403, 1:500); anti-NANOG (Abcam, ab21624, 1:250); anti-nestin (Millipore, MAB5326, 1:500); anti-NeuN (Millipore, ABN78 1:400); anti-OCT4 (Santa Cruz Biotechnology, sc-5279, 1:100); anti-Nucleolin (Abcam, ab22758, 1:200); anti-PAX6 (Covance, PRB-278P, 1:500); anti-SMA (Sigma-Aldrich, A5228, 1:100); anti-SM22 (Abcam, ab14106, 1:200); anti-SOX2 (Santa Cruz Biotechnology, sc-17320, 1:100); anti-Tuj1 (Sigma-Aldrich, T2200, 1:500); Alexa Fluor 555-conjugated wheat germ agglutinin (Thermo Fisher, W32464, 1:500). Other reagents, including deoxycytidine, hydroxyurea, NAC, nocodazole, and thymidine, were purchased from Sigma-Aldrich.

For phenotyping of NPCs by immunofluorescent staining, spheres were dissociated into single cells using 0.25% trypsin and fixed with 4% PFA in PBS. The cells were permeabilized with PBS containing 0.1% Triton X-100 and incubated with anti-GFP (Abcam Ltd), anti-NESTIN (Millipore, Billerica, MA), anti-TUJ1 (SIGMA-Aldrich), anti-MAP2 (Abcam Ltd), anti-CD133 (Thermo Fisher Scientific), anti-NANOG, anti-GFAP (Agilent Technologies, Santa Clara, CA), anti-CNPase (Abcam Ltd), or anti-PCNA (Abcam Ltd.) antibodies. The cells were subsequently incubated with Alexa 488-anti-rabbit IgG, Alexa 488 or Alexa 594-anti-mouse (Abcam Ltd.), Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA), or Alexa 594-conjugated anti-rat IgG (Abcam Ltd.). Images were obtained by confocal microscopy, and the cells were counted manually in a blinded manner.

The slides containing brain organoid cryosections were washed twice with PBS (1x) and blocked with 0,1% TBST with 0.1% BSA diluted in PBS (1x). The following primary antibodies, diluted in 1% BSA, were incubated for 4 h at RT: anti-NESTIN (1:200, mouse—Millipore, Burlington, MA, USA), anti-SOX-2 (1:500, mouse—Biolegend, San Diego, CA, USA), anti-TBR1 (1:200, rabbit—Sigma Prestige, St. Louis, MO, USA), anti-β Tubulin III (1:1000, mouse—Promega, Madison, WI, USA), anti-DCX (1:1000, rabbit—Abcam, Cambridge, UK), anti-MAP-2 (1:500, mouse—Abcam), anti-GFAP (1:200, mouse—DAKO, Glostrup, Denmark), and anti-cleaved caspase-3 (1:100, rabbit—Millipore). The cryosections slides were washed twice with PBS (1X). The following secondary antibodies (1:1000, all from ThermoFisher) diluted in BSA (1%) were incubated for 2 h at RT: AlexaFluor 488 goat anti-mouse and AlexaFluor 594 goat anti-rabbit. The cryosections slides were washed twice with PBS (1x), followed by immunostaining with DAPI (1:1000) diluted in PBS (1x) for 30 min at RT. The cryosections slides were washed twice with PBS (1x), mounted with a mounting media, and stored at 4 °C.