Ab4461

For WB, the detailed procedures were performed as described previously [8] using the following primary antibodies: anti-MCM2 (Abcam, ab4461), anti-MCM3 (Abcam, ab128923), anti-MCM4 (Abcam, ab4459), anti-MCM5 (Abcam, ab75975), anti-MCM6 (Abcam, ab201683), anti-MCM7 (Abcam, ab2360), anti-CDK9 (Abcam, ab76320), anti-CyclinD1 (Abcam, ab16663), anti-CyclinE1 (Abcam, ab33911), anti-p53 (Abcam, ab241566), anti-p21 (Abcam, ab109502), anti-p27 (Abcam, ab32034), and anti-GAPDH (Abcam, ab181602).
For IHC, the standard method was described previously.14 (link) Slides were incubated with primary antibodies (Abs) against MCM2 (Immuway, YM6642), MCM3 (Abcam, ab128923), MCM4 (Immuway, YT2681), MCM5 (Abcam, ab75975), MCM6 (Abcam, ab190948), and MCM7 (Abcam, ab2360).

Immunostaining analyses were carried out on 16 μm brain coronal cryosection. The harvested samples were immersed in MOPS/EGTA/Magnesium Sulfate/Formaldehyde Buffer (MEMFA) overnight at 4°C, then rinsed with 1× PBS and cryopreserved in 30% sucrose in 1× PBS at 4°C. Samples were snap-frozen in Tissue-Tek (4583, Sakura, Torrance, CA, USA) and stored at −80°C. Cryosections were preserved at −20°C.
For BrdU staining, sections were treated with 2M HCl at 37°C for 10 min to denature the DNA. They were then washed five times with 1× PBS for 20 minutes each and blocked with 5% donkey serum in 1× PBS/0.3% Triton X-100. Afterward, immunofluorescence staining was performed following the standard protocol. The following primary antibodies were used against the corresponding antigens: SOX2 (1:500, ab97959, Abcam, Cambridge, UK), CDC42(1:200, sc-8401, Santa cruz, Santa cruz, CA, USA), NEUN (1:500, MAB377, Sigma, St. Louis, MO, USA), TUJ1 (1:500, MAB1195, R&D system, Minneapolis, MN, USA), ZO-1 (1:500, 33-9100, Invitrogen, Waltham, MA, USA), BrdU (1:500, MA3-071, Thermo Fisher Scientific, Waltham, MA, USA), MCM2 (1:300, ab4461, Abcam, Cambridge, UK), and TNC (1:100, MT1, DSHB, Iowa, IA, USA).