Donkey anti mouse alexa fluor 594

Briefly, brain slices and N27 cells were incubated in a blocking solution (2% FBS and 0.3% Triton X-100 in PBS) for 2 h at room temperature. Brain slices were incubated with the primary antibodies in a blocking solution for 2 overnights (mouse anti-CtBP1,1:1000, BD Biosciences, cat no. 612042; mouse anti-CtBP2, 1:1000, BD Biosciences, cat no. 612044; rat anti-CD11b, 1:1000, Serotec; rabbit anti-GFAP, 1:200, DAKO; rabbit anti-NeuN, 1:500, Cell Signaling; rabbit anti-TH, 1:1000, Santa Cruz Biotechnology), while N27 cells were incubated for 1 overnight (mouse anti-CtBP1 and mouse anti-CtBP2, 1:200) at 4 °C. Afterward, tissue and cells were incubated for 2 h at room temperature with the following appropriated secondary antibodies: Alexa Fluor 594 donkey anti-mouse (Abcam), Alexa Fluor 488 donkey anti-rat (Life Technologies) and Alexa Fluor 647 donkey anti-rabbit (Life Technologies) (1:1000 for tissues and 1:200 for cells). Lastly, sections were rinsed with PBS and mounted in Fluoroshield Mounting Medium (Abcam). Images were acquired under the magnification of 40 × using a Zeiss inverted confocal microscopy (Axiobserver Z1, Zeiss).

Twenty millimeters of frozen sections from PF and AF, prefrontal cortex (bregma 4.7 to 3.2 mm), and hypothalamic arcuate nucleus (ARC) (bregma − 1.8 to −4.3) were used for these studies. Sections were fixed with 4% paraformaldehyde and stained overnight at 4 °C with mouse anti-γH2Ax (EMD Millipore, Billerica, MA; 1/250) antibodies. The secondary antibody used was Alexa Fluor 594 donkey anti-mouse (Abcam, Cambridge, MA; 1/500). Sections were mounted with a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) to counterstain nuclei (Invitrogen, Waltham, MA). γH2Ax foci were quantified using ITCN (Image-based Tool for Counting Nuclei for ImageJ, NIH, Bethesda).

Cell cultures were fixed with 2% paraformaldehyde (PFA; Sigma), washed with PBS and blocked with a solution of 3% BSA and 0.1% Tween 20 (Sigma) for 20 min at room temperature to prevent nonspecific binding. Cells were incubated with the primary antibody for 30 min at room temperature followed by overnight incubation at 4 °C. Incubation with the respective secondary antibody was performed at room temperature for 2 h. The antibodies used were rabbit anti-Argonaute-2 (Ago2) (1:200; Cell Signaling, MA, USA), rabbit anti-neuropilin-1 (NRP1) (1:200; BD Biosciences), mouse anti-CD31 (Novocastra, Leica Biosystems, Nussloch GmbH, Germany), rabbit anti-VEGFR2 (Abcam, UK), rabbit anti-ionized calcium-binding adaptor molecule-1 (Iba-1) (1:500; FujiFilm Wako Chemicals, VA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:500; BD Biosciences), Alexa Fluor 546 donkey anti-rabbit, Alexa Fluor 488 donkey anti-mouse, Alexa Fluor 488 donkey anti-rabbit (all 1:200; Life Technologies) and Alexa Fluor 594 donkey anti-mouse (1:200; Abcam). The nuclei were stained with Hoechst 33342 (4 μg/ml; Molecular Probes, OR, USA). Cell preparations were mounted in Dakocytomation fluorescent medium (Dakocytomation Inc., CA, USA) and images were acquired with AxioImager A1 microscope (Carl Zeiss, Gottingen, Germany).