Chicken anti gfp

After being deeply anesthetized with Ketamine and Xylazine, mice were perfused with phosphate buffer saline (PBS) and 4% paraformaldehyde in PBS. Brains were post-fixed overnight with 4%A PFA/PBS, embedded in 2% agarose /PBS, and then sectioned at 50 μm with a vibratome (Leica, Buffalo Grove, IL VT1000S). The following antibodies were used for immunohistochemistry: anti-GFP rabbit (Life Technologies, Thermo Fisher, Waltham, MA A-11122), anti-GFP chicken (Aves labs, Tigard, OR GFP-1020), anti-HA rat (Roche diagnostics, Indianapolis, IN clone 3F10). Whole slide images were taken with a microscope with 5x objective and XY-stage controlled by μManger (https://micro-manager.org). Grid/Collection stitching Fiji plugin (Preibisch et al., 2009 (link)) was used for image assembly. We followed the dual-color in situ protocol described in BraInSitu web site (http://www.nibb.ac.jp/brish/indexE.html) (Watakabe et al., 2006 (link)).

Polyclonal anti-A2BP1(Rbfox1) and anti-Sept11 were generated as described [20 (link), 26 (link)]. Polyclonal rabbit antibodies for green fluorescent protein (GFP) and red fluorescent protein (RFP) were from MBL (Nagoya, Japan) and Rockland Immunochemicals (Gilbertsville, PA), respectively. Anti-GFP (Chicken) was purchased from AVES Labs (Tigard, OR). Rabbit polyclonal anti-Ki67 was from Thermo Scientific Japan (Yokohama, Japan).

The following primary antibodies were used: rabbit anti-atypical PKC (1:1000) (Santa Cruz Biotechnology), rabbit anti-Asense (Cheng-Yu Lee, Univ. of Michigan, Ann Arbor, MI), rat anti-Dpn (1:100) (Abcam), guinea pig anti-Dpn (1:2000) (Jim Skeath, Washington Univ., St Louis, MO), mouse anti-Elav (1:50) (9F8A9, DSHB), chicken anti-GFP (1:500) (Aves Labs), mouse anti-Lamin (ADL67.10, DSHB), guinea pig anti-Miranda (1:500) (Doe lab), rabbit anti-PH3 (EMD Millipore), mouse anti-Pros purified antibody (1:500) (MR1A, Abcam), and rat anti-Worniu (1:100) (Abcam). Fluorophore-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). Texas Red^™-X Phalloidin (ThermoFisher Scientific) and DAPI (Sigma-Aldrich) were applied after secondary antibody staining by following manufacturer’s protocol. Larvae were raised at 29°C until third instar, and the brains were dissected, fixed and stained by following published procedures (Lai and Doe, 2014 ; Lai et al., 2012 (link)). Images were taken with Zeiss confocal microscope LSM710, processed with open source software Fiji (Schindelin et al., 2012 (link)) and assembled in the software Adobe Photoshop or Illustrator (Adobe Systems Inc., San Jose, CA). All statistical analyses in the figures used one-tail t-test included in Microsoft Excel (Microsoft Corp., Redmond, WA).

Cultured cells were lysed in RIPA buffer on ice for 10 min. After 10-min centrifugation at 4 °C, 20,000 × g, whole cell lysates were mixed with 4X LDS sample buffer (Invitrogen) and denatured at 95 °C for 5 min. Samples were loaded on a 4–12% Bis-Tris SDS-PAGE gel, run at 200 V for 45 min in MOPS buffer, and transferred onto a nitrocellulose membrane (Bio-Rad) in an XCell II Blot module (Invitrogen) (15 V, 45 min). After 1-h blocking with 5% nonfat dry milk in PBST, the membrane was incubated with primary antibodies (rabbit anti-UPF1, Cell Signaling Technology #12040; mouse anti-G3BP1, Millipore #05–1938; mouse anti-puromycin clone 12D10, Millipore #MABE343; rabbit anti-hnRNP H, Bethyl Laboratories #A300-511A; chicken anti-GFP, Aves Labs #1010; rabbit anti-phospho-UPF1, Millipore #07-1016; mouse anti-β-Actin, Cell Signaling Technology #4967; rabbit anti-G3BP2, Bethyl Laboratories #A302-040A; rabbit anti-BFP, Evrogen #AB233) diluted (1:2000) in 5% milk/PBST at 4 °C with slow shaking overnight. After incubation, membranes were rinsed three times with PBST, and incubated with IR680- or IR800-conjugated secondary antibodies (Li-Cor) diluted (1:10,000) in 5% milk/PBST at room temperature for 1 h. After three rinses with PBST, membranes were imaged using an Odyssey CLx system (Li-Cor).