Mouse anti tuj1

Sections were washed three times with 1× PBS and then incubated with primary antibodies at 4 °C overnight, after 1 h blocking by 5% normal goat serum (NGS, Sigma, Darmstadt, Germany) and 0.2% Triton X-100 (Sigma, Darmstadt, Germany). The sections were then incubated at room temperature for 2 h with fluorescence conjugated secondary antibodies (Invitrogen, Waltham, MA, USA) and washed with 0.01 M PBS (1×) 3 times before being observed under a confocal laser scanning microscope (Zeiss, LSM800, Jena, Germany). Fluorescence immunohistochemistry was performed by using the following primary antibodies: mouse anti-NF160 (Abcam, 1:400, Cambridge, UK), rabbit anti-NeuN (Abcam, 1:500, Cambridge, UK), chicken anti-Map2 (Abcam, 1:500, Cambridge, UK), rabbit anti-Gfap (Dako, 1:1000, Carpinteria, CA, USA), rabbit anti-Sox2 (Abcam, 1:200, Cambridge, UK), chicken anti-Gfap (Abcam, 1:1000, Cambridge, UK), guinea-pig anti-Iba1 (Synaptic Systems, 1:800, Göttingen, Germany), mouse anti-Tuj1 (R&D, 1:400, Minneapolis, MN, USA), and mouse anti Nestin (Abcam, 1:1000, Cambridge, UK).

Slides with sectioned spheroids were rehydrated and washed three times with PBST (PBS with 0.1% Triton X-100). The slides were then incubated in blocking solution containing 5% donkey serum for 1 h, followed by incubation with primary antibodies diluted with the blocking buffer overnight at 4 °C. Slides were washed three times in PBST for 10 min each and incubated with secondary antibodies diluted in blocking buffer for 1 h at room temperature, washed three more times for 10 min each, and cover slipped with ProLong^TM Gold Antifade Mountant with DAPI (cat. P36931, ThermoFisher, Waltham, MA, USA).
The following antibodies were used: rabbit anti-CC3 (1:750, cat. 9661-s, Cell Signaling, Danvers, MA, USA); rat anti-GFAP (1:1000, cat. 13-0300, ThermoFisher); rat anti-CTIP (1:500, cat. ab18465, Abcam, Cambridge, UK); mouse anti-SATB2 (1:100, cat. ab51502, Abcam); rabbit anti-TBR1 (1:250, cat. Ab31940, Abcam); mouse anti-Ki67 (1:50, cat. 550609, BD Pharmingen, San Diego, CA, USA); rabbit anti-MAP2 (1:1000, cat. PA5-85755, Life Technologies); goat anti-Sox2 (cat. AF2018, R&D Systems) mouse anti-TUJ1 (1:250, cat. ab14545, Abcam); mouse anti-CC1 (1:150, cat. ab16794, Abcam) and 4G8 (1:1000, cat. 800701, Biolegend, San Diego, CA, USA) and PHF13 (1:200, cat. 829001, Biolegend). All secondary antibodies were Alexa Fluor conjugated (Life Technologies) and were used at the concentration 1:500.