3D culture of MCF-10A cells was performed as described (Debnath et al., 2003 (link)). Briefly, 104 cells were seeded onto one chamber of a 4-well chamber slide pre-coated with Matrigel (BD Biosciences). After 31 days, cells were fixed using 4% paraformaldehyde (Fisher) and permeabilized using 0.5% Triton X-100, and sequentially incubated with anti-Laminin-5 (D4B5, EMD Millipore), Alexa Fluor 568 conjugated goat anti-mouse IgG (Invitrogen), anti-GFP (ab13970, Abcam), Alexa Fluor 488 conjugated goat anti-chicken IgG (Invitrogen), and nuclear stain 4,6-diamidino-2-phenylindole (DAPI, Sigma). Images were acquired using the Leica TCS SP5-II Confocal Upright and the PerkinElmer UltraVIEW ERS confocal microscopes at the Molecular Cytology Core Facility at MSKCC and analyzed by MetaMorph software (Molecular Devices). To measure ROS levels, 3D culture of MCF-10A cells after 6-8 days were incubated with Hank's Balanced Salt Solution (HBSS, Invitrogen) containing 5 μM dihydroethidium (DHE, Invitrogen) and 3 μg/ml Hoechst 33342 (Invitrogen) at 37°C for 30 minutes in a humidified 5% CO2 incubator, washed once with HBSS, then immediately imaged.
Anti gfp ab13970
Manufactured by Abcam
Sourced in United Kingdom, Denmark
Anti-GFP (ab13970) is a rabbit monoclonal antibody that recognizes green fluorescent protein (GFP). This antibody can be used to detect and quantify GFP expression in various applications.
Automatically generated - may contain errors
Lab products found in correlation
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Dihydroethidium dhe, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated goat anti chicken igg, by Thermo Fisher Scientific (1 mentions)
Ultraview ers, by PerkinElmer (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Metamorph software, by Molecular Devices (1 mentions)
Matrigel, by BD (1 mentions)
Hrp conjugated antibody, by Agilent Technologies (1 mentions)
Anti perm 3141, by Cell Signaling Technology (1 mentions)
Anti acetylated α tubulin clone 6 11b 1, by Merck Group (1 mentions)
Anti clic5 b 23 sc 133468, by Santa Cruz Biotechnology (1 mentions)
Anti γtubulin clone gtu 88, by Merck Group (1 mentions)
Anti erm, by Cell Signaling Technology (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Alexa fluor 546, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
6 protocols using anti gfp ab13970
1
3D Culture and ROS Imaging of MCF-10A Cells
2
Immunofluorescence and Immunoblotting of Zebrafish
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Zebrafish whole-mount immunofluorescence (IF) was performed as previously described33 (link). The following antibodies were used for IF: anti-CLIC5 (B-23; sc-133468; Santa Cruz Biotechnology; 1:10), anti-CLIC5 (A-11; sc-271863; Santa Cruz Biotechnology; 1:10), anti-γTubulin (clone GTU‐88; Sigma Aldrich; 1:5000), anti-GFP (ab13970; Abcam; 1:200), anti-acetylated αTubulin (clone 6-11B-1; Sigma Aldrich; 1:500) and anti-phospho-Ezrin (Thr567) (PA5-37763; Invitrogen; 1:200). Cy3 (1:1000) and Alexa-488/-546/-647 (1:1000) labelled secondary antibodies were purchased from Jackson Immunoresearch and Molecular Probes (Invitrogen), respectively. Immunoblotting (IB) was performed as previously described47 (link). The following antibodies were used for IB: anti-CLIC5 (B-23; sc-133468; Santa Cruz Biotechnology; 1:200), anti‐pERM (#3141; Cell Signaling; 1:1000), anti‐ERM (#3142; Cell Signaling; 1:1000), anti‐γTubulin (clone GTU‐88; Sigma Aldrich; 1:5000) and respective HRP‐conjugated antibodies (DAKO; 1:5000). Histological and transverse sectioning procedures were performed as previously described33 (link).
3
Immunofluorescence Staining of Zebrafish Larvae
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Larvae were fixed in 4% PFA then stored at −20°C in 100% methanol. Larvae were re‐hydrated, washed in PBS‐T before permeabilization with proteinase K (4333793, Sigma) at 37°C. For A4.1025 antibody staining, larvae were instead permeabilized in 0.25% Trypsin for 15 min on ice. Larvae were then blocked in 5% horse serum and incubated with primary antibodies overnight at 4°C, unless otherwise stated (anti‐myosin (clone A4.1025) (sc‐53088, 1:500, Insight Biotech, UK); anti‐GFP (ab13970, 1:200, Abcam); anti‐mCherry (M11217, 1:100, Invitrogen); anti‐col2a1 (II‐II6B3‐s, 1:20, DSHB, IA, USA); anti‐colXa1 (SAB4200800, 1:100, 48 h incubation needed, Sigma); anti‐sox9a (GTX128370, 1:300, Genetex, CA, USA)). Larvae were washed extensively before incubation with Alexa‐Fluor secondary antibodies (Invitrogen) diluted in 5% horse serum at 1:500 for 2 h at room temperature in the dark. For DAPI staining, larvae incubated in PBS‐T containing DAPI (1 µg/ml) for 1 h at room temperature before washing.
4
Immunofluorescence Staining of Muscle Tissues
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For immunofluorescence staining of cells, primary antibodies used were: anti-MyHC MF-20 (Development Studies Hybridoma Bank); anti-Dys MANDRA17 (Development Studies Hybridoma Bank); anti-GFP (Ab-13970 Abcam); Secondary antibodies used were: anti-mouse Alexa Fluor 546 and anti-chicken Alexa Fluor 488 (Abcam). Nuclei were stained with DAPI (D9542 Sigma).
For immunofluorescence staining of injected and not injected TA muscles. Tissues were dissected, frozen in liquid nitrogen-cooled isopentane and cut on cryostat (LEICA CM 1850, Leica, Germany) into transverse and longitudinal cryostat sections. Primary antibodies used, in addition to those mentioned above, were: anti-dystrophin DMD (Sigma), anti α-sarcoglycan (HPA 007537 Atlas antibodies), anti-nNOS (PA3-032A R&D System) and anti-LAM A/C (MA3-1000 Invitrogen). Secondary antibodies used were: anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 546 (Abcam). Nuclei were stained with DAPI (D9542 Sigma).
For immunofluorescence staining of injected and not injected TA muscles. Tissues were dissected, frozen in liquid nitrogen-cooled isopentane and cut on cryostat (LEICA CM 1850, Leica, Germany) into transverse and longitudinal cryostat sections. Primary antibodies used, in addition to those mentioned above, were: anti-dystrophin DMD (Sigma), anti α-sarcoglycan (HPA 007537 Atlas antibodies), anti-nNOS (PA3-032A R&D System) and anti-LAM A/C (MA3-1000 Invitrogen). Secondary antibodies used were: anti-mouse Alexa Fluor 488, anti-rabbit Alexa Fluor 546 (Abcam). Nuclei were stained with DAPI (D9542 Sigma).
5
Immunohistochemical Analysis of CaMKII and GFP Expression in Rat Brains
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Rats were perfused transcardially with 1X Phosphate Buffered Saline (PBS) and 4% Paraformaldehyde (PFA). The brains were extracted and placed in 4% PFA overnight after which they were placed in varying concentrations of sucrose. Frozen brains were sectioned at a thickness of 50μm and slices were placed in 4C in PBS. Slices were washed in PBST and a blocking solution added (donkey serum, Sigma-Aldrich, St. Louis, MO). Slices were then incubated overnight with the primary antibodies (1:500, Anti-CamKII, ab52476, and Anti-GFP, ab13970, Abcam, Boston, MA). The following day, slices were washed and incubated with the secondary antibodies (1:500, Donkey Anti-Rabbit conjugated with Alexa Fluor 657, and Donkey Anti-Chicken conjugated with Alexa Fluor 488, Jackson ImmunoResearch Labs, West Grove, PA). Images were acquired using the Biotek Cytation 5 Cell Imaging Multi-Mode Reader (Biotek Instruments, Inc., Winooski, VT) configured on an inverted microscope.
6
Immunofluorescence Staining of Muscle Cells
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Muscle fibers and tissue sections were first fixed in 4% paraformaldehyde (PFA) and blocked in the blocking buffer containing PBS, 5% horse serum, 2% BSA, 0.2% Triton X-100 and 0.1% sodium azide for 60 minutes. Then the fibers and sections were incubated with primary antibodies (anti-Pax7, Developmental Studies Hybridoma Bank, University of Iowa; anti-MyoD, M-318, Santa Cruz Biotechnology; anti-β-galactosidase, MAB1802, Millipore Corporation; anti-MyoG, F5D, Santa Cruz Biotechnology; anti-GFP, ab13970, Abcam) diluted in blocking buffer overnight at 4°C, then incubated with secondary antibodies (goat anti-mouse Alexa Fluor 568, A21043, Invitrogen; goat anti-rabbit Alexa Fluor 488, #111-545-144, Jackson ImmunoResearch) and 4′,6-diamidino-2-phenylindole (DAPI) diluted in PBS for 30 minutes at room temperature and mounted with Dako fluorescent mounting medium (Glostrup, Denmark). Fluorescence pictures were taken with a Coolsnap HQ CCD camera (Photometrics, USA) driven by IP Lab software (Scanalytics, USA) in a Leica DMI 6000B fluorescence microscope (Mannheim, Germany). As the analysis of the immunofluorescence was qualitative, identical image handling and fluorescence scoring criteria were applied in all the experiments.
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