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Anti-GFP

Manufactured by Aves Labs
Sourced in United States

The Anti-GFP product from Aves Labs is a monoclonal antibody specifically designed to bind and detect the green fluorescent protein (GFP). This antibody can be used in various applications, such as western blotting, immunohistochemistry, and immunoprecipitation, to identify and quantify the presence of GFP in biological samples.

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28 protocols using Anti-GFP

To measure cell cycle occupancy, we stained replicate RetCFP/+ and RetCFP/CFP whole GI tracts from both male and female mice at E14.5 for phosphohistone H3 (Anti-H3S10p; Millipore Sigma #06-570), an immunomarker specific to cells undergoing mitosis (85 ), and CFP protein (Anti-GFP; Aves labs #GFP-1010) using the immunofluorescence assay described above. Confocal stack images were acquired on the Nikon Eclipse Ti confocal microscope with a 40× oil objective and type A immersion oil and quantified using a custom CellProfiler (86 (link), 87 (link)) workflow. A detailed protocol is provided in SI Appendix.
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Antibodies used for flow cytometry were AF700-anti-mouse Sca-1 (Thermo, 56-5981-82), PerCP/Cy5.5-anti-mouse CD11b (BD Biosciences, 550933), PerCP/Cy5.5-anti-mouse CD31 (BD Biosciences, 562861), PerCP/Cy5.5-anti-mouse CD45 (BD Biosciences, 550944), FITC anti-mouse CD34 (BD Biosciences, 553733), APC-anti-Integrin a7+ (R&D, FAB3518A).
Antibodies used for western blots were Myogenin (F50D) (Santa Cruz, SC-12732), Flag-Tag (3B9) mAb (Abmart, M20008), GAPDH (14C10) Rabbit mAb (Cell Signaling, #2118), Goat anti-mouse IgG-HRP (Santa Cruz, SC-2005), Goat anti-rabbit IgG-HRP (Santa Cruz, SC-2004).
Antibodies used for immunofluorescence staining were anti-MyoD (Santa Cruz, sc-377460), anti-Pax7 (DHSB, RRID:AB_528428), anti-Myh3 (DHSB, F1.625), anti-MyHC (Millipore, 05-716), anti-Laminin (Abcam, ab11575), anti-GFP (Aves Labs, GFP-1010).
Antibodies used for ChIP assays were anti-H3K4me1 (Abcam, ab8895), anti-H3K27ac (Abcam, ab4729), HA antibody (generated by our lab), mouse IgG (Abmart, B30010M), rabbit IgG (Abmart, B30011M).
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Anti-Rbfox1(A2BP1) was produced by ourselves as previously described28 (link). The following mouse monoclonal antibodies were used; anti-Tau-1 (MAB3420; Chemicon International, Temecula, CA), anti-Myc 9E10 and anti-MAP2 (M4403; Sigma-Aldrich, St Louis, MO). Polyclonal rabbit antibodies used were anti-GFP (#598; MBL, Nagoya, Japan), anti-RFP (#600-401-379; Rockland Immunochemicals, Gilbertsville, PA), anti-Rbfox2 (Fox2) (A300-864A-T, Bethyl Laboratories, Montgomery, TX) and anti-Sept1129 (link). anti-GFP (GFP-1020; Chicken) was purchased from AVES Labs (Tigard, OR).
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Imaginal eye and wing discs were dissected from wandering L3 larvae as previously described [8 (link),27 (link)]. Briefly, imaginal discs were fixed in 4% paraformaldehyde and permeabilized in 0.3% PBST prior to staining with antibodies. Antibodies from Developmental Studies Hybridoma Bank: anti-Ptc (mouse, 1:40), anti-Elav (rat 1:800) and anti-Ci (rat, 1:100) were used to stain eye and wing discs. Other antibodies used were anti-phospho-Smad1/5 (pMad) (rabbit 1:100, Cell Signalling), anti-DIAP1 (mouse, 1:50) [28 (link)], and anti-GFP (chicken, 1:100, Aves Labs). Imaginal eye and wing discs were visualized on a compound fluorescent microscope or confocal microscope using the Zen microscopy software (Zeiss microscopy).
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Immunofluorescence studies were conducted on cryosections of adult zebrafish hearts. These hearts were cryoprotected, mounted, sectioned and stained as performed previously40 (link). The following primary antibodies were used: anti-MHC/MF20 (mouse, Developmental Studies Hybridoma Bank, 1:100); anti-α–actinin (mouse, Diagnostic BioSystems, 1:100); anti-Raldh2 (rabbit, Abmart, 1:100); and anti-GFP (chicken, Aves Labs, 1:200). The following secondary antibodies were used: anti-mouse IgG-Alexa 405 (goat, Life technologies, 1:200), anti-mouse IgG-Alexa 594 (goat, Life Technologies, 1:200), anti-rabbit IgGAlexa 568 (goat, Life Technologies, 1:200) and anti-chicken IgG-Alexa 488 (goat, Life Technologies, 1:200). Alexa Fluor 594 conjugated wheat germ agglutinin (WGA) (Life Technologies, 50μg/ml) was used to stain the extracellular matrix. DAPI (1 μg/ml) staining was used to identify nuclei. Notably, we discovered that eGFP from the Tg(Tp1:eGFP)um14 transgene perdured for a longer period in the ventricular outer myocardial wall (Extended Data Fig. 3) compared to d2GFP from the Tg(Tp1:d2GFP)mw43 transgene (Extended Data Fig. 1).
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Embryos or kidneys were fixed overnight in 4% paraformaldehyde (PFA) at 4°C with gentle agitation. After being cryoprotected in 30% sucrose, the specimens were embedded in OCT and cryosectioned at 10 μm thickness. Sections were washed three times in 0.1% Triton-X/PBS for 5 min each wash and blocked for a minimum of 1 hr at room temperature in 5% FBS/0.1% Triton-X/PBS. Sections were incubated in primary antibody at 4 degrees overnight followed by 3 washes in 0.1% Triton-X/PBS. Sections were then incubated in secondary antibody for 2 hr at room temperature, washed 3 times in PBS, mounted with Vectashield, and examined by scanning laser confocal microscopy (Zeiss LSM-510). The primary antibodies used are anti-GFP (chicken; 1:1000, AvesLabs), anti-E-cadherin (Mouse, 1:500, BD Biosciences, CA), anti-Six2 (Rabbit, 1:250, ProteinTech), anti-aPKC (rabbit; 1:500, Santa Cruz, CA), anti-DBA (Biotinylated; 1:500, VectorLabs, Burlingame, CA), anti-LTL (Biotinylated, 1:500, VectorLabs, Burlingame, CA). Nuclei were stained with Topro-3 iodide (1:1,000, Invitrogen). Results shown are representative examples from one of at least three different stainings of three different embryos. We used either Cre positive;EF1a+/+ or Cre negative; EF1aCrb3-GFP/+ tissues as controls.
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We conducted immunofluorescence on ovaries following the protocol described in.74 (link) We stained ovaries with anti-FLAG (1:3000, M2, Sigma Aldrich, St. Louis, MO) and anti-γH2Av (1:1000, a gift from R. S. Hawley). We mounted ovaries with ProLong Gold Antifade Reagent with DAPI (Thermo Fisher Scientific, Waltham, MA). We imaged slides at 63X magnification on a Leica TCS SP8 Four Channel Spectral Confocal System. For each experiment, we used the same imaging parameters across genotypes.
We conducted immunofluorescence on embryos collected in a 0–70 minute window from mh[mel], mh[sim], or mh1 females crossed to males homozygous for P{gcid.EGFP.cid}III.275 (link), a gift from K. McKim. We followed the protocol described in76 (link) to fix and stain the embryos with anti-GFP (1:1000, Aves Labs, Tigard, OR). We mounted and imaged the embryos as described above.
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Dissected mouse intestinal tissues were cut open along the longitude axis, washed in cold PBS, and fixed overnight in 4% paraformaldehyde at 4°C before paraffin-embedding. Antigen retrieval was performed using retriever in buffer A (Electron Microscopy Sciences), and tissue sections were incubated overnight at 4°C with anti-5mC (Active Motif), anti-5hmC (Active Motif), anti-Ki67 (BD Pharmingen), anti-Axin2 (Abcam), anti-Sox9 (Millipore), anti-c-Myc (Santa Cruz Biotechnology), and anti-GFP (Aves Labs) antibodies. After incubation with secondary antibodies (Vector Laboratories) for 2 h at room temperature, samples were mounted in fluorescent mounting medium (Dako) or developed using the VectaStain Elite ABC kit (Vector Laboratories). Images were acquired using a Nikon Eclipse 80i fluorescence microscope and a Leica SP8 confocal microscope.
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Drugs employed in this study were carmustine (BCNU Nitrumon, Sintesa S.A., Bruxelles, Belgium), indinavir sulfate (IDV, Sigma-Aldrich, Milan, Italy), phlorizin (PHZ, Sigma-Aldrich), ritonavir (RTV, Sigma-Aldrich), and temozolomide (TMZ, Sigma-Aldrich). BCNU was resuspended in absolute ethanol; IDV in pure water; PHZ, RTV, and TMZ in DMSO (Sigma-Aldrich) and used at the indicated concentrations. Aphidicolin (Sigma-Aldrich) was used to treat U87MG cells at 1-μM concentration for 24 hours to block cell growth. Antibodies employed and dilutions were the following: anti-actin (1:5000, cat. A4700 Sigma-Aldrich), anti-AMPKα F6 (1:2000, cat. 2793 Cell Signaling Technology, Danvers, MA), anti–phospho-AMPKα Thr172 40H9 (1:1000, cat. 2535 Cell Signaling Technology), anti-GLUT1/SLC2A1 (1:10,000, cat. 07-1401 Merck-Millipore, Vimodrone, MI, Italy), anti–Golgin-97 CDF4 (1:3000, cat. A-21270, Thermo Fisher Scientific, Waltham, MA), anti-GFP (1:700, chicken polyclonal, Aves Labs, Tigard, OR). Species-specific secondary antibodies labeled with peroxidase (Thermo Fisher Scientific) or with Alexa Fluor 488 (1:500, Jackson ImmunoResearch, West Grove, PA) were employed, respectively, for Western blot or immunohistochemistry.
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10

Passive Clearing and Immunostaining of Substantia Nigra

Passive clarification was conducted similarly to protocol established by Tomer et al. 2014 [20 (link)]. In brief, brains were embedded in hydrogel (4% acrylamide/0.05% Bis-acrylamide) and sectioned at 400um thickness on a cryo-microtome. Sections were placed in clearing solution (4% sodium dodecyl sulfate/200mM boric acid, pH 8.5) for 7 days at 37°C and 35 rpm. Sections of clarified substantia nigra region were then selected for immunostaining. Clearing solution was removed by washing 3 times with TBS and then sections were incubated for immunostaining with chicken polyclonal anti-tyrosine hydroxylase (1:200; Abcam Cat# ab76442), rabbit polyclonal anti-GFAP (1:200; DAKO Cat# Z0334), anti-GFP (1:200; AvesLabs) or anti-mCherry (1:200; Abcam Cat# ab167453). Antibodies were diluted in TBS at 37°C and incubated with tissue sections with an orbital shaker at 35 rpm overnight and then at 4°C for 1 day afterward. Multiple washes were performed over 2 days at 37°C (with orbital shaking at 35 rpm) and then cleared sections were placed in TBS containing goat anti-chicken AlexaFluor 647 (1:200; Life Technologies) and donkey anti-rabbit AlexaFluor 555 (1:200; Life Technologies) at 37°C overnight with orbital shaking (35 rpm). The next day, sections were washed several times with TBS and stored at 4°C until imaged.
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