Mab5326

Immunofluorescence staining was performed in vitro on coverslip cultures or frozen brain tissue sections as previously described [27 (link)]. Briefly, cells were fixed in 4% PFA for 20 min (frozen tissue sections did not require this step), washed with phosphate-buffered saline (PBS), and then blocked by QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime, P0260) for 30 min at room temperature. The primary antibodies were added and incubated overnight at 4°C. Primary antibodies included mouse anti-Nestin (Millipore, MAB5326, 1 : 1000), rabbit anti-PAX6 (BioLegend, 901301, 1 : 1000), goat anti-SOX1 (R&D Systems, AF3369, 1 : 1000), rat anti-HOXB4 (DSHB, RRID: AB-2119288, 1 : 100), rabbit anti-Foxg1 (Abcam, Ab18259, 1 : 500), mouse anti-NKX2.1 (Millipore, MAB5460, 1 : 500), rabbit anti-GABA (Sigma-Aldrich, A2052, 1 : 1000), goat anti-CHAT (Millipore, AB144P, 1 : 300), mouse anti-neuronal class III β-tubulin (Tuj1; BioLegend, 801201, 1 : 2000), mouse anti-microtubule-associated protein-2 (Map2; Sigma-Aldrich, M1406, 1 : 1000), mouse anti-human nuclei (HuNu; Millipore, MAB1281, 1 : 500), goat anti-mCherry (Biorbyt, RRID: AB-2687829), and rabbit anti-NeuN (Millipore, ABN78, 1 : 1000). The corresponding fluorescent-conjugated secondary antibodies were applied for 1 h, and the nuclei were stained with DAPI (Boster, AR1177).

Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, blocked 20 min in KPBS (10 mM phosphate buffer, 150 mM NaCl, and 3.4 mM KCl) supplemented with 0.5% BSA, 0.1% Triton X-100, and 5% normal swine serum (Jackson ImmunoResearch, 014-000-121) and incubated overnight at 4°C in blocking buffer containing primary antibody (Oct-4 1:500 Stemgent 09–0023, Nestin 1:5000 Millipore MAB5326, Pax6 1:1000 BioLegend 901301, Ki67 1:200 Millipore MAB4190, βIII-Tubulin 1:200 GeneTex GTX631836, total tau 1:1000 Dako A0024, synapsin-I 1:200 Abcam ab64581, GFAP 1:2000 Dako z0334, GABA 1:1000 Sigma A2052, Doublecortin 1:200 Abcam ab18723, pS129 α-synuclein 1:500 Abcam ab51253, HA-tag 1:1000 Santa Cruz sc-7392, MAP2 1:5000 Abcam ab5392). Subsequently, they were incubated with secondary antibodies (Alexa Fluor Secondary Antibodies from Invitrogen) and Hoechst 34580 (BDbiosciences 565877) for 1 hour at room temperature, mounted in Prolong Gold Antifade Mountant (Thermo Scientific, P36934), and visualized on Leica DM5500 B fluorescent microscope (Leica, DE) or using a Nikon Eclipse Ti 4.10 microscope combined with a Yokogawa CSU-X1 spinning disk confocal scanner and a Hamamatsu UltraVIEW VoX C9100-50 EMCCD camera.