Upon euthanasia, half of each tumor was embedded in OCT and snap-frozen on dry ice. Tumors were then sectioned, fixed in 4% PFA, and incubated overnight at 4°C with primary anti-GFP antibody at 1:250 dilution (rabbit, polyclonal, Abcam). Then, the sections were stained with anti-rabbit secondary antibodies conjugated with Alexa 488 (1:2,000 dilution). The sections were imaged using a Zeiss LSM 510 confocal microscope and analyzed with Zen (Zeiss) software.
Primary anti gfp antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The Primary anti-GFP antibody is a laboratory reagent used to detect and identify the green fluorescent protein (GFP) in biological samples. It is a specific, high-affinity antibody raised against the GFP protein. The antibody can be used in various immunodetection techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to visualize and quantify the presence of GFP in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (6 mentions)
Lsm 900 confocal microscope, by Zeiss (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti actin, by EASYBIO (1 mentions)
510 meta laser scanning confocal microscope, by Zeiss (1 mentions)
Secondary goat anti rabbit igg hrp, by Abcam (1 mentions)
Horseradish peroxidase, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Abcam (1 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (1 mentions)
Donkey anti chicken alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Aminoethylcarbazole, by Agilent Technologies (1 mentions)
Vecastain abc kit, by Vector Laboratories (1 mentions)
Photomicroscope, by Olympus (1 mentions)
Hrp conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Irdye 800cw goat anti rabbit igg secondary antibody, by LI COR (1 mentions)
Protease inhibitor, by Roche (1 mentions)
19 protocols using primary anti gfp antibody
1
Immunohistochemical Analysis of Tumor GFP
2
Quantifying GFP-ELF3 Protein Levels
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Seeds of 35S::GFP:ELF3 (Col-0) were surface sterilized, plated onto 1× MS plates with 0.8% w/v agar, 0.25% w/v sucrose, and 0.5 g/L MES with a pH of 5.7. Seeds were subsequently stratified for 3 days before being transferred to a short-day growth cabinet with 125 µmol of WL and a constant temperature of 22°C. Seedlings were subsequently grown for 17 days before light treatments (as described above) were started at ZT7 on Day 18. The seedlings were thoroughly grinded and total proteins were extracted with the buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40 (v/v), 1 mM PMSF, 1 mM DTT, 2 mg/mL Chymostatin, 2 mg/mL Leupeptin, 2 mg/mL Pepstatin, 2 mg/mL Aprotinin, 50 mM MG132, 50 mM MG115, 50 mM ALLN, 2 mM NaF, and 2 mM Na3VO4. About 10 µL GFP tagged ELF3 proteins (added with SDS loading buffer) were separated by 8% SDS-PAGE. Immunoblotting was performed using a 1:2,000 dilution of the primary anti-GFP antibody (Abcam, Cambridge, UK; ab6556) or a 1:2,000 dilution of the primary anti-Actin (EASYBIO, Seoul, Korea). The 1:3,000 horseradish peroxidase (HRP)-linked anti-rabbit IgG was used as secondary antibodies. Experiments were repeated twice, with similar results observed on each occasion.
3
Quantifying Mutant and Non-Mutant Protein Expression
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To determine the relative expression of mutant and non-mutant CRYBB2 and CRYAA in transfected cells, quantitative real-time PCR (RT-qPCR) was performed following the protocol described by Wang et al.59 (link). Efficiency was determined before performing qPCR, and only primers with an efficiency above 95% were used. Endogenous GAPDH was used for normalization of the threshold cycle (Ct) value detected for both the transfected and normal cells. Western blotting was also carried out to verify expression of the proteins in the transfected cells. Cells transfected with GFP served as controls. Target proteins were blotted using a primary anti-GFP antibody (1:20000, Abcam, USA) and horseradish peroxidase (HRP) Goat anti-Rabbit IgG antibody (1:20000, Boster, USA). Protein expression was normalized to that of GAPDH.
4
Visualizing Bone Osteogenesis in Zebrafish
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Paraffin sections from Tg(sp7:EGFP)b1212 transgenic skulls at age 12 wpf (21 mm SL) were collected for immunohistochemistry. The primary anti-GFP antibody (Abcam) diluted to 1:500 and secondary Alexa Fluor 488 Donkey Anti-rabbit IgG diluted to 1:500 were used to detect the GFP reporter per manufacturer’s recommendations. DAPI counterstaining allowed for nuclei visualization. Specimens were observed using Zeiss 510 META Confocal Laser Scanning Microscope and 488 nm and 405 nm laser lines.
5
Detecting SPL10-GFP Fusion Protein
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Fresh Arabidopsis leaves (0.1 g) were homogenized in 0.2 ml of protein extraction buffer (0.125 mM Tris, pH6.8, 4% w/v SDS, 18% glycerol, 0.024% w/v bromophenol-blue, 1.43 M β-mercaptoethanol, 0.2% protease inhibitor). After boiling for 10 min, the insoluble fraction was removed by centrifugation, and the supernatant (denatured protein) was separated on a 12% SDS PAGE gel and transferred onto a nitrocellulose membrane, followed by incubation with primary anti-GFP antibody (Abcam, ab290, Cambridge, MA, USA) and secondary goat anti-rabbit IgG HRP (Abcam) antibody. The membrane was developed with Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The expression of SPL10-GFP fusion protein was also investigated using a Biological Confocal Laser Scanning Microscope FV10-ASW, and the emission wavelength for GFP and DAPI channels are 488 nm and 405 nm, respectively (OLYMPUS, Tokyo, Japan).
6
Western Blot Analysis of 35S:SPL12m-GFP
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Fresh leaves of 30-day-old plants of WT and 35S:SPL12m-GFP alfalfa were homogenized in 0.2 ml of protein extraction buffer (0.125 mM Tris, pH 6.8, 4% w/v SDS, 18% glycerol, 0.024% w/v bromophenol-blue, 1.43 M β-mercaptoethanol, 0.2% protease inhibitor). After centrifugation at 15,000 g for 10 min the insoluble fraction (pellet) was discarded, and the denatured proteins were separated on a 12% SDS PAGE gel. Separated proteins were then transferred onto a nitrocellulose membrane, followed by incubation with primary anti-GFP antibody (Abcam, ab290, Cambridge, MA) and secondary horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (Abcam) antibody. The signals were developed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA).
7
Quantifying mdg1 RNA in Ovarian Egg Chambers
8
GFP Immunostaining in Brain Slices
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After electrophysiological recordings, brain slices were fixed in 4% paraformaldehyde overnight. Washing was done with a step-wise protocol using a tris-buffered saline, and 0.3% triton solution. Slices were transferred to the same tris-buffered saline containing donkey serum (5%) for 2 h to block unspecific binding of antibodies. Incubation with a primary anti-GFP antibody (1:1000; polyclonal chicken, Abcam) in 5% donkey serum was done for 72 h at 4°C. Subsequently, slices were rinsed with tris-buffered saline and incubated with donkey-anti-chicken-Alexa Fluor 488 (1:500; Invitrogen) and Alexa Fluor 568 conjugated Streptavidin (1:1000; MoBiTec) for 72 h at 4°C. After the final rinsing, slices were mounted with ProLong Gold Antifade (Invitrogen), and imaged using a Zeiss LSM900 confocal microscope (Oberkochen, Germany). Image analysis and processing was done using the Zeiss ZEN software and ImageJ freeware1 .
9
Quantifying mdg1 RNA in Ovarian Egg Chambers
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mdg1 RNA FISH on ovaries was performed as described 56 (link) using CAL Fluor Red 590-labeled Stellaris oligo probes (Table S11 ). After the RNA FISH protocol, egg chambers were blocked with SBX (1% BSA; 0.1% Triton X-100; 2xSSC) for 30 min and then incubated with primary anti-GFP antibody (Abcam) for 24h at 4°C. After 3x washing (10 min with SBX), samples were incubated with fluorescent secondary antibody for 12h at 4°C. Stacks of soma nuclei were imaged on a Zeiss LSM780 confocal microscope and a maximum intensity projection of 3 slices was generated.
10
Histopathological Evaluation of Transplanted BM-MSCs
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The entire colon was removed from the cecum to the anus, fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 4-μm sections. After hematoxylin and eosin (H&E) staining, histological analysis was performed in a blind manner. The following three parameters were studied as described previously:22 (link) inflammation severity (0=none, 1=mild, 2=moderate, and 3=severe), inflammation extent (0=none, 1=mucosa, 2=mucosal and submucosa, and 3=transmural), and crypt damage (0=none, 1=basal 1/3 damaged, 2=basal 2/3 damaged, 3=crypts loss, but surface epithelium present, 4=both crypts and surface epithelium loss). Total histological score was defined as the sum of the three parameters.
Immunohistochemical (IHC) staining for GFP, using a Vecastain ABC kit (Vector Laboratories, Burlingame, CA, USA), was performed to identify the in vivo localization of transplanted BM-MSCs in the inflamed colon. Colonic tissue samples were incubated first with the primary anti-GFP antibody (Abcam, Cambridge, MA, USA) overnight at 4°C, then with a biotinylated secondary linking antibody, and finally with a streptavidin-peroxidase complex for 1 hour. The final color product was developed using aminoethylcarbazole (Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin, and tissues were photographed using an Olympus photomicroscope (Olympus Corp., Tokyo, Japan).
Immunohistochemical (IHC) staining for GFP, using a Vecastain ABC kit (Vector Laboratories, Burlingame, CA, USA), was performed to identify the in vivo localization of transplanted BM-MSCs in the inflamed colon. Colonic tissue samples were incubated first with the primary anti-GFP antibody (Abcam, Cambridge, MA, USA) overnight at 4°C, then with a biotinylated secondary linking antibody, and finally with a streptavidin-peroxidase complex for 1 hour. The final color product was developed using aminoethylcarbazole (Dako, Glostrup, Denmark). Sections were counterstained with hematoxylin, and tissues were photographed using an Olympus photomicroscope (Olympus Corp., Tokyo, Japan).
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