Mice were anesthetized with an overdose of rodent cocktail (a combination of ketamine 50 mg/kg, xylazine 5mg/kg, acepromazine 1mg/kg injected i.p.) and perfused transcardially with 0.9% saline, followed by 4% PFA in PBS (pH 7.4). Brains were immersed in 30% sucrose at 4°C until sunk and flash frozen with cold 2-methylbutane (Fisher Scientific, Hampton, NH, USA). Coronal sections of the pregenual mPFC were obtained at 35 μm using a cryostat (Leica CM3050 S, Concord, ON, Canada). For double-labeled immunofluorescence, endogenous mouse antibodies were blocked using a mouse Ig blocking reagent (Vector Laboratories, Burlingame, USA). mPFC sections were incubated overnight at 4°C with mouse monoclonal anti-SMI-32 antibody (1:1000 dilution, Covance, Burlington, NC, USA, Cat. #14941802) and Goat anti-GFP antibody (1:1000 dilution, Novus Biologicals, Oakville, ON, Canada, Cat. #NB100–1770). Immunostaining was visualized with Alexa 488-conjugated (Jackson Immunoresearch, Van Allen Way, Carlsbad, CA, USA, Cat. #705–545-003), and Alexa Fluor 555-conjugated (Life technologies, Toronto, ON, Canada, Cat. #A21429) secondary antibodies raised in donkey.
Goat anti gfp antibody
Manufactured by Novus Biologicals
Sourced in Canada
The Goat anti-GFP antibody is a primary antibody that recognizes and binds to the green fluorescent protein (GFP) molecule. This antibody can be used in various immunological techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and visualize the presence of GFP-tagged proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cm3050, by Leica (2 mentions)
Mouse ig blocking reagent, by Vector Laboratories (2 mentions)
2 methylbutane, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti smi 32 antibody, by Fortrea (2 mentions)
Lead citrate, by Merck Group (1 mentions)
Toluidine blue, by Merck Group (1 mentions)
Uranyl acetate, by Merck Group (1 mentions)
Jem 100cx, by JEOL (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Nembutal, by Abbott (1 mentions)
3 protocols using goat anti gfp antibody
1
Double-Labeled Immunofluorescence of mPFC
2
Double-Labeled Immunofluorescence of mPFC
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Mice were anesthetized with an overdose of rodent cocktail (a combination of ketamine 50 mg/kg, xylazine 5mg/kg, acepromazine 1mg/kg injected i.p.) and perfused transcardially with 0.9% saline, followed by 4% PFA in PBS (pH 7.4). Brains were immersed in 30% sucrose at 4°C until sunk and flash frozen with cold 2-methylbutane (Fisher Scientific, Hampton, NH, USA). Coronal sections of the pregenual mPFC were obtained at 35 μm using a cryostat (Leica CM3050 S, Concord, ON, Canada). For double-labeled immunofluorescence, endogenous mouse antibodies were blocked using a mouse Ig blocking reagent (Vector Laboratories, Burlingame, USA). mPFC sections were incubated overnight at 4°C with mouse monoclonal anti-SMI-32 antibody (1:1000 dilution, Covance, Burlington, NC, USA, Cat. #14941802) and Goat anti-GFP antibody (1:1000 dilution, Novus Biologicals, Oakville, ON, Canada, Cat. #NB100–1770). Immunostaining was visualized with Alexa 488-conjugated (Jackson Immunoresearch, Van Allen Way, Carlsbad, CA, USA, Cat. #705–545-003), and Alexa Fluor 555-conjugated (Life technologies, Toronto, ON, Canada, Cat. #A21429) secondary antibodies raised in donkey.
3
Ultrastructural Analysis of Transplanted Neural Progenitor Cells
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To assess the spatial organization of the perivascular cell clusters, an electron microscopy analysis of GFP-positive cells was carried out. A week after FGF-2 (GFP)-transduced NPC transplantation, the animals were deeply anesthetized with Nembutal (Abbott, North Chicago, IL, USA) and transcardially perfused with 0.9% saline followed by 2% paraformaldehyde (Sigma-Aldrich) and 0.2% glutaraldehyde (Sigma-Aldrich). Brains were then washed in 0.1 M of PBS. Horizontal 150-µm sections were cut on a vibratome.
The sections were incubated in goat anti-GFP antibody (1:500; Novus Biologicals, LLC). A biotinylated secondary anti-goat antibody (1:200; DakoCytomation) was used for visualization of the primary antibody. Subsequently, the avidin-biotin complex (ABC) method was carried out, and the peroxidase reaction was developed in 3,3¢-diaminobenzidine (DAB; Sigma-Aldrich).
Immunostained sections were postfixed in 1% OsO 4 (Sigma-Aldrich), dehydrated in a graded series of ethanol, and embedded in Epon 812 (Fluka, Buchs, Switzerland). Semithin sections were cut with a glass knife and stained lightly with 1% toluidine blue (Sigma-Aldrich). For the identification of individual GFP-labeled cells, ultrathin sections were cut with a diamond knife, stained with uranyl acetate (Sigma-Aldrich) and lead citrate (Sigma-Aldrich), and examined with an electron microscope JEM-100CX (JEOL, Tokyo, Japan).
The sections were incubated in goat anti-GFP antibody (1:500; Novus Biologicals, LLC). A biotinylated secondary anti-goat antibody (1:200; DakoCytomation) was used for visualization of the primary antibody. Subsequently, the avidin-biotin complex (ABC) method was carried out, and the peroxidase reaction was developed in 3,3¢-diaminobenzidine (DAB; Sigma-Aldrich).
Immunostained sections were postfixed in 1% OsO 4 (Sigma-Aldrich), dehydrated in a graded series of ethanol, and embedded in Epon 812 (Fluka, Buchs, Switzerland). Semithin sections were cut with a glass knife and stained lightly with 1% toluidine blue (Sigma-Aldrich). For the identification of individual GFP-labeled cells, ultrathin sections were cut with a diamond knife, stained with uranyl acetate (Sigma-Aldrich) and lead citrate (Sigma-Aldrich), and examined with an electron microscope JEM-100CX (JEOL, Tokyo, Japan).
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