Prb 278p

Cells were fixed in 4% PFA for 15 min and then permeabilized with 0.3% Triton X-100 in PBS for 10 min. Then, cells were blocked with 10% Normal Goat Serum for 1 hr and incubated in the appropriate primary antibodies from mouse or rabbit: Sox2 (1:1000, Abcam), Oct4 (1:250, Santa Cruz, Sc-5279), Nestin (1:500, R&D Systems, MAB1259), Pax6 (1:300, Covance, PRB-278P), β-III tubulin (TuJ1, 1:200 Covance, MMS-435P), Tau (1:500, Santa Cruz, Sc-5587), O4 (R&D Systems, MAB1326 1:500), GalC (Santa Cruz 1:250 sc-518055), and Glial fibrillary acidic protein (GFAP, 1:1000, Dako, G9269). Then, cells were washed three times for 5 min with 1× PBS and incubated 1 hr in appropriate secondary antibody at 1:1000. The secondary antibodies used were red (594 Alexa Fluor, Thermo Fisher Scientific, Invitrogen, A-11032, A-11037) or green (488 Alexa Fluor, Thermo Fisher Scientific, Invitrogen, A-11001, A-11070) goat-anti-mouse antibodies or goat-anti-rabbit antibodies. Cells were visualized on fluorescent microscope.

LG sections, monolayer, and 3D cultures were stained by using an indirect sequential immunoenzymatic technique. The following primary antibodies were used for immunostaining: rabbit polyclonal antibody to Pax6 (PRB‐278P, Covance, Princeton, NJ,
http://www.covance.com), mouse monoclonal E‐cadherin antibody (clone 36/E‐cadherin; catalog no. 610181, BD Biosciences, San Jose, CA,
http://www.bdbiosciences.com; or clone DECMA‐1; EMD Millipore, Billerica, MA,
http://www.emdmillipore.com), rat monoclonal antibody against Ser‐28 phosphohistone‐H3 (P‐H3) (clone HTA28; catalog no. H9908, Sigma‐Aldrich, St. Louis, MO,
http://www.sigmaaldrich.com), laminin antibody produced in rabbit (catalog no. L 9393; Sigma‐Aldrich), and cytokeratin 5 (Krt5) rabbit polyclonal antibody (catalog no. AF138; Covance), mouse monoclonal α‐smooth muscle actin antibody (clone 1A4; catalog no. A2547, Sigma‐Aldrich), rat monoclonal antibody to platelet endothelial cell adhesion molecule (CD31) (clone MEC 13.3; catalog no. 557355, BD Biosciences), goat polyclonal antibody to thrombospondin‐1 (c‐20; catalog no. sc‐7653; Santa Cruz Biotechnology Inc., Santa Cruz, CA,
http://www.scbt.com). Complimentary secondary antibodies were obtained from Thermo Fisher. Images were taken by using a Zeiss LSM 780 laser scanning confocal microscope (Zeiss, Stuttgart, Germany,
http://www.zeiss.com).

The sections were dewaxed, completely rehydrated and for antigen retrieval boiled in sodium citrate 0.1 M pH 6. The sections were washed in PBS-T and incubated in PBS-T with 1.5% H₂O₂ for 30 min to inactivate endogenous peroxidase. After inactivation, tissue was washed in PBT and blocked 1 h in PBS-T with 0.1% albumin bovine serum (BSA, A2153, Sigma) and 10% lysine 1 M (L5626, Sigma). Next, sections were incubated overnight at room temperature in PBS-T with 0.1% BSA and 0.01% sodium azide (S2002, Sigma) with αNKX2.2 (1:5, raised in mouse, Hybridoma Bank, 74.5A5) or αPAX6 (1:200, raised in rabbit, Covance, PRB-278P). The day after, the tissue was rinsed in PBS-T and incubated 1 h with αMouse (1:200, raised in goat, Vector Labs, BA-2020) or αRabbit (1:200, raised in goat, Vector Labs, BA-1000) biotinylated secondary antibody. Afterwards, the sections were washed in PBS-T and incubated in PBS-T with Avidin–Biotin Complex (1:500, Vectastain PK-4000) for 1 h. Finally, tissue was washed in PBS-T and Tris 0.1 M pH 7 and the immunolabeling was revealed in Tris 0.1 M with 1% 3-3′ diaminobenzidine tetrahydroc (DAB, Acros Organics W0572M) and 0.003% H₂O₂ leading to a brown precipitate (0.025% Ammonium Nickel Sulphate was added to obtain black precipitate).

Embryonic mouse cortices were homogenized in sucrose solution (0.32 M sucrose, 5 mM Mg(Ac)₂, 5 mM CaCl₂, 50 mM HEPES pH 8, 1 mM DTT, 0.1% Triton X-100, and 0.1 mM EDTA), fixed in 37% formaldehyde, and then treated with 1.25 M glycine. The samples were then centrifuged, and the pellet (i.e., nuclei) was washed with Nelson buffer. Chromatin fragments were prepared, immunoprecipitated, and analyzed, as previously described [28 (link),38 (link)].
We tested the following antibodies for KAT2a ChIP (NBP1-00845, Novus Biologicals; ab1831, Abcam; 07-1545, Millipore, MA, USA; 3305, Cell Signaling; sc-20698 (H-75), Santa Cruz; 607201 Biolegend) [39 (link)], Pax6 ChIP (PRB-278P, Covance; AB2237, Chemicon), and BAF155 ChIP (sc-10756, Santa Cruz; ab126180, Abcam), but none of the antibodies showed any difference from the IgG control and were, therefore, considered not suitable for ChIP experiments.

Cerebral organoids were fixed with 1% PFA in 120 mM phosphate buffer pH 7.4 for 20 min at room temperature and subjected to cryosectioning (14 µm) and immunofluorescence as described (Camp et al., 2015 (link)). The following primary antibodies were used: rabbit anti-PAX6 (PRB-278P; Covance), sheep anti-TBR2 (AF6166; R+D systems), rat anti-CTIP2 (ab18465; Abcam), rabbit anti-KI67 (ab15580; Abcam). The secondary antibodies, used in combination with DAPI staining, were all donkey-derived and conjugated with Alexa 488, 555 or 647 (Life Technologies). Images were acquired with a Zeiss LSM 880 Airy inverted microscope, using 10X (0.45 NA) and 20X (0.8 NA) Plan-Apochromat objectives, and analysed using Fiji. Quantifications were carried out in cortical regions of D28 and D52-54 cerebral organoids by counting, from the ventricular to the pial surface, either all PAX6 and TBR2 positive and negative nuclei stained by DAPI in 50 μm and 100 μm wide fields, respectively, or all KI67-positive cells in 100 μm wide fields. An average of 350 cells per sample were counted. Statistical significance was calculated using the Mann–Whitney U-test.