Rabbit anti gfp

Ten microliter translation reactions were performed with 2.8 nM mEGFP mRNA (folded) and 1 μM WT NT-hFMRP as described previously with nLuc mRNA. Forty microliters of 2× reducing SDS sample buffer (Bio-Rad # 1610737) was then added and heated at 70 °C for 15 min. Ten microliters was then separated by standard Tris-Glycine SDS-PAGE (Thermo # XP04200BOX) and transferred on to 0.2 μm polyvinylidene difluoride membrane (Thermo # 88520). Membranes were then blocked with 5% (w/v) nonfat dry milk in TBST (1× Tris-buffered saline with 0.1% (v/v) Tween 20) for 30 min at RT before overnight incubation with primary antibodies in TBST at 4 °C. After three 10 min washes with TBST, membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibody in TBST for 1 h at RT and then washed again with three 10 min washes with TBST. Chemiluminescence was performed with SuperSignal West Pico PLUS for GFP (Thermo # 34577) and with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo # 34095) for tubulin. Blots were imaged using an Azure Sapphire Biomolecular Imager. Rabbit anti-GFP (Cell Signaling # 2956S) was used at 1:1000. Mouse antitubulin (Sigma # T9026-2ML) was used at 1:1000. HRP-conjugated goat anti-rabbit IgG (H + L) (Thermo # 31460) was used at 1:60,000 for GFP and HRP-conjugated goat antimouse IgG (H + L) (Thermo # 31430) was used at 1:10,000 for tubulin.

Cells were washed twice in PBS and total cell proteins were lyse with 1× Laemmli buffer. Proteins were separated on 12.5% SDS–PAGE gels, followed by western blotting according to standard procedures. Nitrocellulose membranes containing protein cell lysate incubated with primary antibodies at a dilution of 1:2000 rabbit anti-GFP (Cell Signaling; 2956) and 1:5000 mouse anti-Beta Actin (Sigma A5441). Proteins were visualized by a chemiluminescence detection kit for horseradish peroxidase (Biological Industries) and quantified using a CCD camera and Image-J software. The uncropped western blot can be found in Supplementary Fig. 4.

Immunohistochemistry was performed according to standard protocols. Briefly, 4μm serial sections were dewaxed, rehydrated and immersed in 3% hydrogen peroxide for 20 minutes to quench endogenous peroxidase activity. Antigen retrieval was carried out at 95°C for 25 minutes in 10 mM Tris, 1 mM EDTA solution, pH 9.0. After cooling, sections were incubated with blocking buffer (PBS supplemented with 5% goat serum and 1% bovine serum albumin) for 1 hour at room temperature (RT). Primary antibodies were diluted in blocking buffer and applied for 1 hour at RT: rabbit anti-p53 (Cell Signaling Technology, 2527) at 1:200 dilution, rabbit anti-GFP (Cell Signaling Technology, 2956) at 1:75 dilution, anti-Cleaved Caspase-3 (Cell Signaling Technology, 9664) at 1:50 dilution and rabbit anti-luciferase (Abcam ab185924) at 1:200 dilution. Sections were then incubated with a biotinylated anti-rabbit secondary antibody at RT for 45 min, followed by incubation with streptavidin-biotin peroxidase solution at RT for 45 min. Visualization of the first antibody binding was carried out using DAB, according to the manufacturer’s instructions (Vector Labs). Finally, sections were lightly counterstained using Mayer’s haematoxylin and allowed to dry before mounting and digitizing using the Hamamatsu Nanozoomer-XR. Pixels positive for p53 and GFP were quantified using Adobe Photoshop.

For detecting dokb-GAL4 expression across larval development in a central brain lobe, appropriately aged larval CNS samples were fixed with 4% paraformaldehyde for 20 min. at RT. The 1° antibody, rabbit anti-GFP (Cell Signaling Technology, #2956), was applied at 1:1000 and the counterstaining antibody, mouse anti-DN-cad (Cell Signaling Technology, #14215), was applied at a 1:5 concentration. Alex Fluor 488 Donkey Anti-Rb IgG (H + L) (Invitrogen, #A32790) and Alex Fluor 555 Donkey Anti-Rb IgG (H + L) (Invitrogen, # A31572) were applied at a 1:500 concentration.
For detecting dokb-GAL4 expression in the larval and adult CNS, samples were fixed in 4% paraformaldehyde for 20 min. at RT. Samples were labelled with rabbit anti-GFP.Alexa 488 conjugate (1:400; Invitrogen, # A21311) and counterstained with primary mouse anti-Brp (nc82) (1:40; DSHB) and secondary donkey anti-mouse Alexa 647 (1:400; Invitrogen, #A31571). Wandering 3rd instar male larvae were used. Adult male flies were aged 3-4 days old.
All images were obtained from a Zeiss LSM880 microscope.

BrdU incorporation assays were performed from 32.5 hpf. Briefly, dechorionated zebrafish embryos were incubated in 15% DMSO fish water containing 10 mM BrdU (Sigma) for 30 minutes at 4°C, and then incubated at 28°C for one hour; embryos were subsequently fixed in PFA at 4°C overnight for immunostaining.
Whole-mount immunostaining was performed following standard protocols as previously described [53] (link) with some modifications. The antibodies used were as follows: mouse anti-BrdU (Sigma), rabbit anti-GFP (Cell Signaling Technology, Inc., MA), Cy2-conjugated anti-mouse IgG, and Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). The nuclei were visualized by staining with DAPI (Sigma) for 5 minutes.

For immunoblotting, cells were seeded in 24 or 96 well plates (Corning) then later harvested by trypsinisation, centrifuged, washed and lysed in SDS buffer (0.35 M Tris pH 6.8, 0.1 g/ml sodium dodecyl sulfate, 93 mg/ml dithiothreitol, 30% glycerol, 50 μg/ml bromophenol blue). Proteins were resolved by SDS-PAGE then electro-blotted onto Immobilion-P membranes (Millipore). Membranes were blocked in 5% dried milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween-20) then incubated overnight at 4°C with the following primary antibodies diluted in milk: rabbit anti-Mcl-1 (Santa-Cruz), sheep anti-Tao1 [60 (link)], mouse anti-Cyclin B1 (Millipore), rabbit anti-Bcl-xL (Cell Signalling), sheep anti-Bub3 (Holland and Taylor, unpublished), rabbit anti-FBW7 (Bethyl), mouse anti-Bak (Calbiochem), rabbit anti-Bax (Santa-Cruz), mouse anti-myc tag (4A6, Millipore), rabbit anti-GFP (Cell Signalling). Following TBST washes, blots were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies (Zymed). Bound secondaries were then detected by addition of EZ-ECL Chemiluminescence Reagent (Biological Industries) or Luminata™ Forte Western HRP Substrate (Millipore) and imaged using a Biospectrum 500 imaging system (UVP).