Guinea pig anti doublecortin

At 35 days after hWJ-MSCs transplantation, the rats were sacrificed and the brains were collected and fixed in 4% formalin. The coronal sections (15 μm thickness) through the area of ischemia were prepared using a cryostat (CM1900; Leica, Heidelberg, Germany). The sections were blocked in 10% goat serum in phosphate-buffered saline/Tween (0.01 M sodium phosphate buffer, pH 7.4, 0.05% Tween 20) for 1 hour at room temperature, and incubated with the primary antibody diluted in blocking buffer at 4°C for 24 hours, followed by incubation with secondary antibody overnight at 4°C. Immunofluorescence signals were observed under a fluorescence microscope (DMIRB; Leica). Primary antibodies were as follows: guinea pig anti-doublecortin (1:1,000; Millipore, Boston, MA, USA), mouse anti-microtubule-associated protein 2 (MAP2) (1:1,000; Millipore) or mouse anti-Tuj1 (1:400; Sigma). Secondary antibodies were as follows: Alexa fluor 488-conjugated goat anti-guinea pig IgG (1:1,000; Invitrogen, Carlsbad, CA, USA) and FITC-conjugated goat anti-mouse IgG (1:1,000; Millipore). After that, sections were counterstained with Hoechst (1:1,000) to indicate cell nuclei.