Goat anti doublecortin sc 8066

Following testing rat’s brains were extracted and immersed in 4% paraformaldehyde for 48 hours. Brains were then transferred to a 10% glycerol solution for 1 day and a 20% glycerol solution for at least 2 days before being sectioned at 40 μm on a freezing sliding microtome. Dorsal hippocampal sections (~4 mm posterior to Bregma) were stained for thymidine kinase to confirm genotypes and dorsal and ventral (~6 mm posterior to Bregma) sections were stained for the immature neuronal marker doublecortin to confirm inhibition of neurogenesis (Fig 1). Immunohistochemistry was performed with goat anti-HSV-1 thymidine kinase (sc-28038; Santa Cruz Biotechnology, USA) and goat anti-doublecortin (sc-8066; Santa Cruz Biotechnology, USA) primary antibodies, diluted in PBS with 0.05% triton-x and 3% normal horse serum, on free-floating sections. Secondary detection was performed with Alexa488-conjugated secondary antibodies (Life Technologies, USA) diluted in PBS. Sections were counterstained with DAPI, mounted onto slides, coverslipped with PVA-DABCO, and imaged with a Leica SP8 confocal microscope. DCX⁺ cell density measurements were calculated by dividing the number of DCX⁺ cells by the granule cell layer volume in one dorsal and one ventral section per animal.

For immunofluorescence, every 6th section was collected and washed in PBS. The sections were then incubated for 2 h in blocking buffer PBS-plus (10% donkey serum, 0.2% Triton-X 100 in PBS) to block unspecific binding sites and to permeabilize the tissues. After the blocking step, the sections were incubated overnight at 4°C with the primary antibodies diluted in incubation buffer (PBS with 3% donkey serum). The following primary antibodies and concentrations were used: rabbit-anti GFAP (Z0334, 1:1000, Dako), goat-anti Doublecortin (sc-8066, 1:250, Santa Cruz Biotechnology), mouse-anti Prox1 (MAB5654, 1:500, Chemicon International), rabbit-anti Ki67 (NCL-Ki67p 1:500, Novocastra), rabbit-anti Tbr2 (23345, 1:800, Abcam), goat-anti Sox2 (sc-17320, 1:200, Santa Cruz Biotechnology), goat-anti NeuroD (sc-1086, 1:400, Santa Cruz), rabbit-anti BLBP (ab32423, 1:400, Abcam). After several washes in PBS, the sections were incubated for 4 h at room temperature in secondary antibodies diluted in incubation buffer. The concentration of secondary antibodies CY3 (711-495-152, Jackson Immuno Research) and CY5 (715-175-15, Jackson Immuno Research) was 1:500. After the incubation, sections were washed several times in PBS and incubated in DAPI (861405, 1:4000, Invitrogen) for 10 min, then washed again in PBS and mounted on slides in Aqua Poly/Mount.