Vectashield hard set anti fade reagent

Coronal sections of the hippocampal region were used for double immunofluorescence labeling with BrdU/doublecortin (DCX), or BrdU/NeuN. DNA was denatured as described above. Sections were incubated with a mixture of anti-BrdU (1:500, AbD Serotec, Kidlington, UK) with rabbit polyclonal anti-DCX (1:1000, Abcam, Cambridge, UK) or polyclonal rabbit anti-NeuN (1:1000, Millipore-Chemicon, Burlington, MA, USA). Sections were then rinsed in PBS, followed by incubation with a mixture of the secondary antibodies Alexa Fluor 488 goat anti-rat IgG (1:500, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 568 goat anti-rabbit IgG (1:500, Molecular Probes, Eugene, OR, USA) for 1h at RT. Sections were rinsed, and nuclei were counter-stained with NucBlue (Molecular probes, Eugene, OR, USA). Sections were mounted and dehydrated. Slides were cover-slipped using Vectashield Hard Set anti-fade reagent (Vector Laboratories, Burlingame, CA, USA) and stored in darkness at 4 °C. For staining against Reelin, sagittal sections were incubated with mouse monoclonal anti-Reelin (1:2000, Abcam, Cambridge, UK) and then with Alexa Fluor 488 goat anti-mouse IgG (1:500, Molecular Probes, Eugene, OR, USA) as secondary antibody.

24 h after seeding, cells on culture plates were fixed with 4% PFA for 1 h and rinsed with PBS. For high-affinity F-actin cytochemistry, the cells were incubated with Alexa Fluor 594-conjugated phalloidin (1:30, Thermo Scientific, CA, USA). For immunocytochemistry the cells were incubated with 10% normal goat serum in PBS-T 0.2% at RT for 1 h, followed by an incubation with the primary antibodies against PSA-NCAM (1:200, Cell Signalling Technologies, Danvers, MA, USA), GFAP (1:500, DAKO; Denmark), DCX (1:1000, Abchem, UK), or 8-OH(d)G (1:250, QED Bioscience, CA, USA) for 12 h at RT. Additionally, a fraction of cells on culture plates were treated with 10 µg/µl DNase I (Qiagen, Germany), or 5 µg/µl RNase (Qiagen, Germany), before incubation with 8-OH(d)G antibody. Culture plates were rinsed in PBS-T 0.2%, followed by incubation with Alexa Fluor 488 or Alexa Fluor 647 conjugated secondary antibody against rabbit IgG (1:500, Molecular Probes, USA) for 1 h at RT. Cell nuclei were counterstained with NucBlue (Thermo Scientific, CA, USA). Culture plates were coverslipped using Vectashield Hard Set anti-fade reagent (Vector Laboratories, CA, USA) and stored in darkness at 4 °C. The integrated density of 8-OH(d)G-Ir in NPCs above cell-free background was analyzed using Image J software.

From each brain of group 1, every twelfth section was used for double immunofluorescence with BrdU/DCX, BrdU/GFAP and for Sirt1 immunofluorescence. From each brain of group 2, every twelfth section was stained for BrdU/NeuN and BrdU/GFAP. For BrdU staining, DNA was denatured as described above. Sections were incubated with the following primary antibodies: rat monoclonal anti BrdU (1:500, AbD Serotec, UK) and rabbit polyclonal anti DCX (1:1000, Abcam, England); polyclonal rabbit anti GFAP (1:2000, DAKO, Denmark), polyclonal rabbit anti NeuN (1:1000, Millipore-Chemicon, CA) or polyclonal rabbit anti Sirtuine-1 (Sirt1, 1:1000, Millipore-Chemicon, CA). Sections were then rinsed in PBS, followed by incubation with the following secondary antibodies: Alexa Fluor 488 goat anti rat IgG (1:500, Molecular Probes, USA) and Alexa Fluor 568 goat anti rabbit IgG (1:500, Molecular Probes, USA) for one hour at room temperature. After washing, nuclei were counterstained with NucBlue Fixed Cell Stain (Molecular probes, USA). Sections were then mounted on slides and dehydrated. Finally slides were coverslipped using Vectashield Hard Set anti-fade reagent (Vector Laboratories, CA) and kept in darkness at 4° C.