Mouse anti gfp

Hippocampal neurons cultured for 3 days were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.4% Triton X-100 in PBS. The samples were incubated with PBS containing 10% donkey serum for 1 h at room temperature and then were incubated with the primary antibodies: rabbit anti-GFP (1:500, Invitrogen), goat anti-TrkB (1:500, R&D, Minneapolis, MN, USA, AF1494), mouse anti-GFP (1:200, Millipore, Temecula, CA, USA, MAB3580), or rabbit anti-JNK3 (1:500, Millipore, 04-893) at 4 °C overnight. Samples were washed three times with PBS and incubated with fluorescent secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) for 1 h at room temperature. All of the images were captured with a Zeiss LSM780 confocal microscope fitted with a × 63 oil-immersion objective lens (Microstructural Platform of Shandong University).

Larvae were fixed in 3.7% formaldehyde at room temperature for 20 min, after which wing imaginal discs were dissected. The following primary antibodies were used: rat anti-DE-Cad (1:50) (Developmental Studies Hybridoma Bank (DSHB)), mouse anti-GFP (1: 5,000 for Western blotting, Millipore), rabbit anti-Myc (1:100, Santa Cruz Biotechnology), mouse anti-β-Galaxtosidase (1:500, Promega), mouse anti-human YAP (1:50), rabbit anti-cleaved-Dcp-1 (1:200, Cell Signaling Technology), mouse anti-human α-Cat (1:50, Santa Cruz Biotechnology), rabbit anti-human Scrib (1:50, Abcam), and rabbit anti-human YAP (1:50, Cell Signaling Technologies).
Secondary antibodies (1:200) were as follows: goat anti-mouse IgG Alexa 488, goat anti-mouse IgG Alexa 568, goat anti-mouse IgG Alexa 647, goat anti-rabbit IgG Alexa 488, goat anti-rabbit IgG Alexa 568, goat anti-rat IgG Alexa 488 and Alexa488-conjugated phalloidin (all from Thermo Fisher Scientific).

The method for cell culturing in a microfluidic chamber has been previously described.^{37 (link)} Briefly, the microfluidic chambers were fixed to poly-D-lysine-coated 60-mm dishes. Suspensions of neurons were plated into the compartment of the cell soma; the other side was filled with neurobasal medium supplemented with B27. After 4 days of culturing, axons grew through the microgrooves and extended into the axonal compartment. Lentivirus was added into the cell body side, and cells were cultured for another 48 h. Then, BDNF (50 ng/ml) was administered in each compartment for 30 min. Cells were then fixed with 4% PFA and stained with mouse anti-GFP (1:500, Millipore) and rabbit anti-pErk5 (1:500, CST, Beverly, MA, USA) antibodies. Pictures were captured with a × 63 oil-immersion objective lens in the Eclipse TE 2000-U inverted fluorescence microscope (Nikon). The fluorescence intensity was measured by NIH ImageJ.

For quantification of specific GFP positive cell populations, the following triple labeling was performed: mature neurons—mouse anti-GFP (1:200; Santa Cruz Biotechnology), chicken anti-β-III tubulin (1:100; Millipore), and rat anti-BrdU (1:400; Accurate Chemical & Scientific Corp.); neuroblasts—mouse anti-GFP, goat anti-doublecortin (DCX; 1:400; Santa Cruz Biotechnology), and rat anti-BrdU; progenitor cells—mouse anti-GFP, rat anti-BrdU, rabbit anti-nestin (1:200; Millipore); stem cells- goat anti-GFP, mouse anti-nestin, rabbit anti-GFAP (1:500; Dako); mature astrocytes—goat anti-GFP, mouse anti-GFAP, rabbit anti-S100ß (1:3000, Abcam). The following secondary antibodies were used from Jackson ImmunoResearch Laboratories (West Grove, PA): biotinylated species-specific anti-IgG (all used at 1:250), Cy3-conjugated Donkey anti-Rat (1:500), Alexa Fluor 647 (AF647)-conjugated Donkey anti-Mouse (1:250), Cy2-conjugated Streptavidin (1:250), DAPI Nucleic Acid Stain (1:10,000, Life Technologies, Grand Island, NY).

Zebrafish embryos from the Tg(fli1a:EGFP)y1 strain at 48 hpf were dechorionated and fixed in 2% PFA in PBS, overnight at 4 °C. Embryos were then washed four times for 5 min in PBST (PBS+0.1% Tween20). Permeabilization in PSBT+0.5% Triton X-100 was performed for 30 min at room temperature. Embryos were then blocked in a solution of PBST+0.5% Triton X-100, 10% normal goat serum and 1% BSA for 2 h at room temperature. Embryos were incubated with primary antibodies in blocking solution overnight at 4 °C. Successively, embryos were washed six times in PBST over 4 h at room temperature and then incubated with secondary antibodies in blocking solution, overnight at 4 °C. Embryos were washed finally six times in PBST over 4 h at room temperature and equilibrated in glycerol 85% in PBS. The following antibodies were used: mouse anti-GFP (1:2,000 Millipore); rabbit anti-podocalyxin (1:150 kindly provided by Heinz-Georg Belting); rabbit anti-mouse Alexa-488-conjugated IgG (1:400); and goat anti-rabbit Alexa 546-conjugated IgG (1:400). For the microscope analysis, we mounted on slides the trunk and tail regions dissected from five to six embryos of each samples. Images were taken with a Leica TCS SP2 confocal microscope, using oil-immersion objective × 40.