Alexa fluor 488 goat anti mouse conjugate

Following fixation, brains were paraffin-embedded and serially sectioned at 4 µm. Three sets of four serial coronal sections every 100 µm were taken at each of the following two levels, as previously described [6 (link)]: level 1 started at the medial septal nucleus and level 2 at the hippocampal formation.
Six slides per brain (three slides per level) were stained with Cresyl Violet (CV; C5042-10G; Sigma- Aldrich, Overijse, Belgium), and two slides per brain (one slide per level) were incubated with each of the following primary antibodies: mouse monoclonal anti-glial fibrillary acidic protein antibody (GFAP) (G6171, Sigma-Aldrich, St Louis, MO, USA), anti-NG2 chondroitin sulfate proteoglycan antibody (MAB5384, Millipore, Billerica, MA, USA), or a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method for fluorescent in situ end labeling of double-stranded DNA fragmentation (Apoptag S7110; Millipore). The secondary antibody was Alexa Fluor® 488 goat anti-mouse conjugate (Invitrogen, Sigma-Aldrich, Bornem, Belgium) or Alexa Fluor® 647 goat anti-mouse conjugate. Sections were counterstained with Hoechst 33342 (Sigma-Aldrich, Bornem, Belgium). The following brain areas were assessed: frontal cortex (FC), corpus callosum (CC), caudate nucleus (CN), internal capsule (IC), putamen (P), and hippocampus (HC).

Brains were microtomed at 4 µm thickness from anterior to posterior. A set of 5 serial coronal sections at 100 µm intervals was taken at each of the following two levels. The first level started at the medial septal nucleus and second at the hippocampal formation. Supplementary Fig. 3 illustrates the regions of interest. Sections were placed onto poly-L- lysine coated slides (Sigma-Aldrich, Bornem, Belgium). Staining was done with Cresyl Violet (C5042-10G, Sigma-Aldrich, Overijse, Belgium) and the primary antibodies used included mouse monoclonal anti-NeuN antibody (MAB377, Millipore, Billerica, MA, USA), anti-NG2 chondroitin sulfate proteoglycan antibody (MAB5384, Millipore, Billerica, MA, USA), mouse monoclonal anti-glial fibrillary acidic protein antibody (GFAP) (G6171, Sigma-Aldrich, St Louis, MO, USA), mouse monoclonal anti-human Ki67 (M724001-2, Agilent, Diegem, Belgium), mouse monoclonal anti-synaptophysin (Sy38, ab8049, Abcam, Cambridge, UK). The secondary antibody was Alexa Fluor® 488 goat anti-mouse conjugate (Invitrogen). Degenerating nuclei were visualized by a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method for fluorescent in situ end labelling of double-stranded DNA fragmentation (Apoptag S7110; Millipore, Billerica, MA, USA). Sections were counterstained with Hoechst 33342 (Sigma-Aldrich, Bornem, Belgium).