Donkey anti rabbit 647

Immunofluorescence staining on mouse brain section was performed as previously described (Tao et al., 2015 (link)). In brief, mice anesthetized by 10% chloral hydrate were perfused intracardiacally with 30 ml normal saline, followed by 4% paraformaldehyde (4% PFA). Then the brains were quickly removed and post-fixed for about 12 h in the 4% PFA and further dehydrated in 30% PB-buffered sucrose solution for 36 h at 4°C. Brain slices were sectioned at 30 μm or 40 μm by a cryostat (Leica) and washed in PBS, blocked in the buffer (10% donkey serum and 0.2% Triton X-100 in PBS) for 90 min, and then incubated in primary antibody diluted in the blocking buffer overnight at 4°C. After being washed with PBS, brain slices were incubated in the secondary antibody diluted in the PBS for 2 h at room temperature, and washed with PBS. The slices were finally mounted with anti-quenching mounting medium (Thermo Fisher Scientific). The primary and secondary antibodies used for immunostaining were as follows: Rabbit anti-GFAP (1:2000, DAKO), Donkey anti-Rabbit 647 (1:1000, Jackson ImmunoResearch), Donkey anti-Rabbit 488 (1:1000, Jackson ImmunoResearch); 4′,6-diamidino-2-phenylindole (DAPI, 1:10,000, Sigma-Aldrich).

To stain with various antibodies, brain sections were incubated for 1 h at room temperature in blocking solution (10% FBS, 0.3% Triton X-100), then in the primary antibody at 4°C overnight. Sections were washed 3×20 min in 0.3% Triton X-100 in PBS, then incubated in Alexa-Fluor-conjugated or DyLight-conjugated secondary antibodies for 2 h at room temperature. Incubation with DAPI for 5 min and 3×20 min 0.1% Triton X-100 washes followed, both at room temperature.
Primary antibodies used in this study were: rabbit polyclonal anti-PRDM16 (1:500; Bjork et al., 2010 (link)) and goat anti-SOX2 (1:150; R&D Systems), mouse anti-TUJ1 (1:1000; Millipore), rabbit anti-Ki67 (1:500; Abcam) and rabbit anti-Caspase-3 (1:200; Cell Signaling). The secondary antibodies used were: donkey anti-rabbit-647 (1:1000; Jackson ImmunoResearch), donkey anti-goat-549 (1:1000; ImmunoResearch) and donkey anti-mouse-647 (1:1000; Jackson ImmunoResearch). Imaging was done on a Leica CTR6500 confocal laser-scanning microscope. Volocity (ImproVision) and Photoshop (Adobe Systems) softwares were used for image processing.

The antibodies for experiments were as follows; CAPS2 (ab69794), TPH2 (ab111828), CAMK2B (ab52476), GFP (ab290; ab13970) (all from Abcam, Cambridge, UK), c-Fos (for WB, NBP2-50,037; Novus biologicals, Briarwood, CO, USA; for IHC, sc-52-G; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-ERK1/2 (p-ERK, Thr202/Tyr204, #9101), ERK1/2 (#9102; Cell Signaling Technology, Danvers, MA, USA), β-actin (SC-47787; Santa Cruz Biotechnology), TH (AB152; EMD Millipore, Temecula, CA, USA). Secondary antibodies used include donkey anti-goat cy3 (Jackson, 1:500), donkey anti-rabbit 647 (Jackson, 1:500), and donkey anti-chick 488 (Invitrogen, 1:500).

For each animal, six 50 μm coronal brain sections (250 μm between sections) were washed four times, for 10 min in Tris-buffered saline pH 7.4 (TBS) at room temperature, blocked for 1 h in TBS containing 3% horse serum (Millipore; S9135) and 0.3% Triton X-100 (Sigma; 9002-93-1) at room temperature, followed by an overnight staining at 4°C in TBS containing 3% horse serum and 0.3% Triton X-100 with the following primary antibodies: rat anti-BrdU (Abcam, ab6326; 1/200), chicken anti-GFP (Aves, GFP-1020; 1/500), mouse anti-S100β (Abcam, ab66028; 1/100) and rabbit anti-Tbr2 (Abcam, ab23345; 1/300). The next day, brain sections were washed four times for 10 min in TBS at room temperature, blocked for 30 min TBS containing 3% horse serum and 0.3% Triton X-100 at room temperature, and stained for 2 h in TBS containing 3% horse serum and 0.3% Triton X-100 at room temperature with the following secondary antibodies: donkey anti-rat 405 (Abcam, ab175670; 1/400), donkey anti-chicken 488 (Jackson ImmunoResearch, 703-545-155; 1/400), donkey anti-mouse 549 (Jackson ImmunoResearch, 715-507-003; 1/400) and donkey anti-rabbit 647 (Jackson ImmunoResearch, 711-605-152; 1/400). Afterwards, sections were washed four times for 10 min in TBS at room temperature and mounted on glass slides.

HC11 cells were fixed using ice-cold MeOH for 10 min. After washing with 1X PBS, cells were incubated for 1 h at room temperature with primary antibodies [anti-γH2AX (SCBT, sc-517348; 1:100), anti-pATR (Genetex, GTX128145; 1:100), anti-PLIN2 (generously provided by Jim McManaman; 1:100)] in a humid incubation chamber. After incubation, cells were washed three times using 1X PBS and incubated for 1 h at room temperature in darkness using corresponding secondary antibodies [donkey anti-rabbit 488 (Jackson ImmunoResearch, 711-546-152; 1:100), donkey anti-rabbit 647 (Jackson ImmunoResearch, 711-606-152; 1:100), donkey anti-mouse 488 (Jackson ImmunoResearch, 715-546-150; 1:100) and/or donkey anti-mouse 647 (Jackson ImmunoResearch, 715-606-150; 1:100)] and Phalloidin-iFluor 488 Reagent (Abcam, ab176753; 1:1000) when indicated. Cells were washed three times using 1X PBS and incubated with Hoechst 33342 (AnaSpec, AS-83218; 1:1000) for 10 min. Cells were mounted using fluoromount-G (Southern Biotech, 0100-01) and visualized using Zeiss Axio Imager Microscope. The integrated density of PLIN2, nuclear γH2AX and nuclear pATR was quantified using ImageJ.