Reporter visualization. Mice were sacrificed by CO2 asphyxiation, and their eyes were isolated and typically fixed in 2% paraformaldehyde in sodium acetate buffer, pH 6.0 [12 (link)]. The corneas surgically dissected from the P0-Cre;R26-tdTomato animals were fixed in 4% paraformaldehyde and examined directly following mounting, without antibody-mediated enhancement. In the case of the MADM mice, whole corneas were incubated overnight at 4 °C in combined primary antibodies (chicken anti-green fluorescent protein [GFP]; Aves Labs, Tigard, OR; 1:500, and goat anti-c-Myc; Novus Biologicals, Littleton, CO; 1:200) diluted in Tris-buffered saline (TBS), pH 7.3, with 1.0% bovine serum albumin (BSA) and 0.4% Triton X-100. Anti-c-Myc antibody was preabsorbed with fixed wild-type tissue before use [13 (link)]. The following day, the slides were labeled serially with fluorescein isothiocyanate (FITC)–conjugated and Alexa Fluor 555–conjugated secondary antibodies (1:200 and 1:400 dilution, respectively) to enhance visualization of GFP and c-Myc-RFP, respectively. After several buffer rinses, four radial incisions were made in each cornea to produce “petals,” and the resulting flatmounts were placed on glass slides with the endothelium facing up and then coverslipped using Vectashield fluorescence mounting medium (Vector Labs, Burlingame, CA) [14 (link)].
Chicken anti green fluorescent protein gfp
Manufactured by Aves Labs
Sourced in United States
Chicken anti-green fluorescent protein [GFP] is a laboratory reagent used for the detection and quantification of green fluorescent protein in biological samples. It is a polyclonal antibody produced in chickens that specifically binds to the GFP protein.
Automatically generated - may contain errors
Lab products found in correlation
Aqua poly mount, by Polysciences (2 mentions)
Goat anti c myc, by Novus Biologicals (1 mentions)
Vectashield fluorescence mounting medium, by Vector Laboratories (1 mentions)
Chicken anti beta galactosidase lacz, by Abcam (1 mentions)
Goat anti phox2b, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti red fluorescent protein rfp, by Rockland Immunochemicals (1 mentions)
Vt1000, by Leica (1 mentions)
4 protocols using chicken anti green fluorescent protein gfp
1
Cornea Visualization and Immunohistochemistry
2
Immunohistochemistry Staining Antibodies
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Chicken anti-beta galactosidase (LacZ) 1:4000 (Abcam, Cambridge, MA), Chicken anti-green fluorescent protein (GFP) 1:1000 (Aves Labs, Tilgard, OR), Lbx1 (1:10,000, gift from C Birchmeier), Rabbit anti-neurokinin 1 receptor (NK1R) 1:2000 (Millipore, Billerica, MA), Goat anti-Phox2b 1:500 (Santa Cruz Biotechnology (SCBT), Santa Cruz, CA), Goat anti-Islet-1 1:500, (Neuromics). All antibodies used have been previously characterized and no signals were present in genetic or antibody controls.
3
Immunofluorescence Staining of Cultured Cells
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Cell cultures were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature. Primary antibodies were diluted in PBS, 0.5% Triton X-100 and 5% normal goat serum. Specimens were incubated overnight at 4°C. After three washes with PBS, cells were incubated with species-specific secondary antibodies conjugated to fluorophores for 2 h at room temperature. Once again, samples were washed with PBS three times. For nuclei staining, cells were incubated for 5 min with 0.1 μg/mL DAPI (4′6′-diamino-2-phenylindone) in PBS 0.1 M. Coverslips were finally mounted onto a glass slide with a mounting medium (Aqua Poly/Mount; Polysciences). The following primary antibodies and dilutions were used: chicken anti-Green Fluorescent Protein (GFP, Aves Labs, 1:1,000), rabbit anti-Red Fluorescent Protein (RFP, Rockland, 1:1,000), mouse anti-major microtubule associated protein (MAP2; Sigma, 1:500), guinea pig polyclonal anti-vesicular GABA transporter (vGAT, Synaptic Systems, 1:200), and polyclonal anti-vesicular glutamate transporter 1 (vGLUT11, Synaptic Systems, 1:1,000).
4
Immunohistochemical Analysis of Zebrafish Brain
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Adult zebrafish were killed with 0.04% (w/v) MS-222 and the brain was excised under a dissection microscope by using forceps and suspended briefly in PBS 24 h after injections. The dissected brain was fixed in 4% (w/v) paraformaldehyde/PBS at 4 °C overnight. The fixed brain was washed twice with PBS and post-fixed in 100% methanol at −20 °C overnight. The brain was then rehydrated through a descending methanol series (75%, 50%, 25%, and 0% methanol in PBS) and embedded in 2% (w/v) low-melting agarose for sectioning. Then, 50-µm thick transverse brain sections were made by a vibratome (VT1000S, Leica Biosystems, Nussloch, Germany). Brain sections were treated with blocking buffer (0.2% (w/v) bovine serum albumin, 1% (v/v) dimethyl sulfoxide, 0.1% (v/v) Tween-20 in PBS) and processed for immunohistochemistry to detect eGFP by using chicken anti-green fluorescent protein (GFP) (1:1000, Aves Labs, USA) as a primary antibody. Anti-chicken Alexa 488-conjugated antibodies (1:1000, Invitrogen, USA) were used as secondary antibody. Processed slices were mounted in aqua polymount (PolyScience, USA) for confocal imaging.
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