To screen MAbs specific to OBs, cells (A549, TGF-β1-treated A549, SAOS-2, U2OS, hMSCs, and hFOB) were dissociated by trypsin-EDTA (Welgene) and filtered through a 40-μm strainer and washed by phosphate-buffered saline (PBS, pH 7.4). Cells were resuspended in ice-cold PBA (1% bovine serum albumin and 0.01% NaN3 in PBS) and incubated with primary antibodies, followed by the incubation with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA), Alexa488-conjugated anti-mouse IgG (Thermo Fisher Scientific, Seoul, Korea), phycoerythrin (PE)-conjugated anti-mouse IgG (Thermo Fisher Scientific), and/or DyLight 650-conjugated anti-mouse IgG (Thermo Fisher Scientific). To analyze only live cells, propidium iodide (PI) (Sigma-Aldrich)-negative cells were gated and analyzed by FACSCalibur (BD biosciences) and Cell Quest Software (BD biosciences). For multicolor flow cytometry, cells were incubated with appropriate primary antibodies for 30 min and incubated with Dylight 649-(Vector Laboratories, Burlingame, CA) or Alexa 488-conjugated anti-mouse IgG (Thermo Fisher Scientific) after osteogenic or adipogenic differentiation of hMSCs. The cells were than incubated with PE-conjugated mouse monoclonal anti-human CD73, CD90, or CD146 antibodies, rabbit polyclonal anti-human CD164, or rabbit polyclonal anti-integrin α2, α3, or αV antibodies.
Alexa 488 conjugated anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Alexa Fluor 488-conjugated anti-mouse IgG is a secondary antibody that binds to mouse immunoglobulin G (IgG) antibodies. The Alexa Fluor 488 dye is covalently attached to the antibody, providing a fluorescent label that can be detected using fluorescence-based techniques, such as flow cytometry and fluorescence microscopy.
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Lab products found in correlation
Alexa 568 conjugated anti rabbit igg, by Thermo Fisher Scientific (13 mentions)
Alexa 594 conjugated anti rabbit igg, by Thermo Fisher Scientific (12 mentions)
Alexa 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (10 mentions)
Lsm 700, by Zeiss (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (7 mentions)
Paraformaldehyde (pfa), by Merck Group (7 mentions)
Vectashield 19 with dapi, by Vector Laboratories (6 mentions)
Hoechst 33342, by Thermo Fisher Scientific (5 mentions)
Alexa 555 conjugated anti rabbit igg, by Thermo Fisher Scientific (4 mentions)
Axio imager m2 microscope, by Zeiss (4 mentions)
Anti p rpa, by Fortis Life Sciences (3 mentions)
Anti γh2ax, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa546 conjugated anti rabbit igg, by Thermo Fisher Scientific (3 mentions)
Axiocam icc1, by Zeiss (3 mentions)
Hrp conjugated anti rabbit igg, by GE Healthcare (3 mentions)
Tissuefaxs 200, by TissueGnostics (2 mentions)
Anti ezrin antibody 3c12, by Santa Cruz Biotechnology (2 mentions)
Zen 2 blue edition software, by Zeiss (2 mentions)
Facscalibur, by BD (2 mentions)
85 protocols using alexa 488 conjugated anti mouse igg
1
Characterizing Cell Surface Markers by Flow Cytometry
2
CXCR6 Knockdown in MKN45-Luc Cells
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MKN45‐Luc cells treated with si‐CXCR6 for 5 days were collected and fixed with 10% (v/v) formalin in PBS. Then cells were washed with PBS and incubated with mouse anti‐human CXCR6 antibody (R&D Systems) at a final concentration of 25 μg/mL, followed by incubation with Alexa488‐conjugated anti‐mouse IgG (Life Technologies, Carlsbad, CA, USA). Flow cytometric analysis was performed using the BD FACSVerse (Becton Dickinson, Franklin Lakes, NJ, USA).
3
Mapping of the 3A6 Epitope
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For mapping of the 3A6 epitope, two VP1 peptides that span the known 5D8/1 epitope19 (link) were used. High-binding enzyme immunoassay plates were coated with the peptides (RPTNSESIPALTAAE or PALTAVETGATNPLV; Genscript, Piscataway, USA and previously described in Richardson19 (link),34 (link) or with CVB3 VLP20 (link) in 50 mM sodium carbonate buffer pH 9.4 overnight. Plates were then blocked with 5% normal goat serum (NGS; Vector) in PBS and incubated for 2 hr with varying dilutions of clone 5D8/1 or 3A6 in 5%NGS/ PBS. The binding of the antibody was detected with Alexa 488-conjugated anti-mouse IgG or anti-rat IgG (Life Technologies; 1/400). Fluorescence was measured using a Pherastar FS plate reader.
4
Immunofluorescence Staining of Neural Progenitor Cells
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Primary NPCs were dispersed by trypsin treatment, plated onto 35-mm dishes or 12-well plates precoated with poly-L-ornithine, and cultured for 24 hrs. Cells were fixed with 10% formalin solution at room temperature for 20 min and permeabilized with methanol at room temperature for 20 min. Fixed cells were incubated with primary antibodies at 4°C overnight, and with secondary antibodies at room temperature for 90 min. The primary antibodies used are: necdin (GN1; 1∶500), Sox2 (R&D Systems; 1∶300), nestin (ST-1; 1∶1000), βIII-tubulin (Promega; 1∶1000), GFAP (1∶1000) [50] (link), BrdU (Abcam; 1∶500), necdin (NC243; 1∶500) [52] (link), Bmi-1 (Merck Millipore; 1∶300), and Green Fluorescent Protein (GFP) (1∶500; MBL). The secondary antibodies are: cyanine 3-conjugated guinea pig IgG (Life Technologies; 1∶500), Alexa 488-conjugated anti-mouse IgG (Life Technologies; 1∶500), Alexa 488-conjugated anti-rabbit IgG (Life Technologies; 1∶500), cyanine 3-conjugated anti-rabbit IgG (Jackson ImmunoResearch; 1∶500), cyanine 3-conjugated anti-rat IgG (Jackson ImmunoResearch; 1∶500), and Alexa 488-conjugated anti-guinea pig IgG (Life Technologies; 1∶500). Chromosomal DNA was stained with 5 µM Hoechst 33342 (Sigma-Aldrich). Immunofluorescence images were observed by fluorescence microscopy and processed using Adobe Photoshop CS5 software.
5
Epidermal Dissection and Immunostaining
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Dissection and immunostaining of larval epidermis were performed as previously described.52 (link) Primary antibodies: anti-Fasciclin III (Developmental Studies Hybridoma Bank, Iowa City, IA, USA, 1:50), anti-activated Caspase-3 (Cell Signaling, Danvers, MA, USA, 1:150), and anti-GFP (Life Technologies, Basel, Switzerland, 1:500). Secondary antibodies: alexa488-conjugated anti-mouse IgG (Life Technologies, 1:1000), Cy3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA, 1:1000). TUNEL labeling kit (Roche, Basel, Switzerland) was used to label apoptotic cells.
6
Antibody Characterization for Stem Cell Research
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Antibodies against CREB (Cat# 9197), GAPDH (Cat# 97166S), GFAP (Cat# 12389), Ki67 (Cat# 9449) p35 (Cat# 2680S), p39 (Cat# 3275S), pCREB (Cat# 9198), SOX2 (Cat# 3579), mouse IgG HRP-linked (Cat# 7076) and rabbit IgG HRP-linked (Cat# 7074) were purchased from Cell Signalling Technology (Danvers, Massachusetts, USA). Alexa488-conjugated anti-mouse IgG (Cat# A11012) and Alex594-conjugated anti-rabbit IgG (Cat# A11012) were purchased from Life Technologies. The antibody against nestin (Cat# MAB1259) was purchased from R&D Signalling Technology (Minneapolis, Minnesota, USA). Antibodies against CDK5 (Cat# sc-6247) and DYRK1A (Cat# sc-100376) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against p-CDK5 (Y15) (SAB450427) and βIII-tubulin (Cat# T8860), BMP4 (Cat# SRP3016) and Doxycycline (Cat# D9891) were purchased from Sigma Aldrich (Saint Louis, MO, USA). ALGERNON was synthesised and characterised in-house as previously reported [42 (link)].
7
Quantification of Nectin-4 Expression
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Cells were washed with phosphate buffered saline (PBS) and trypsinized in 0.025% trypsin/0.24 mM ethylenediaminetetraacetic acid (EDTA). They were pelleted by centrifugation at 3,300 × g for 1 min, and then were resuspended in Hank's balanced salt solution (HBSS; Life Technologies) containing 2% FCS and incubated on ice for 30 min with anti-human Nectin-4 monoclonal antibody (Clone 337516, R&D Systems, Minneapolis, MN, USA), anti-human SLAM antibody [A12 (7D4); BioLegend, San Diego, CA, USA], and anti-CD46 antibody (M177; HyCult Biotech, Uden, The Netherlands). Next, the cells were washed in PBS containing 2% FCS, and incubated on ice for a further 30 min with Alexa 488-conjugated anti-mouse IgG (Life Technologies). Finally, the cells were washed with PBS containing 2% FCS, and the intensity of fluorescence was measured by a FACSCalibur (BD Biosciences, San Jose, CA, USA). To derive the relative level of Nectin-4 expression, the mean fluorescent intensity (MFI) of cells stained with anti-Nectin-4 antibody was calculated using Flowjo software ver 9.7.5 (TreeStar, San Carlos, CA).
8
3D Culture and Immunofluorescence Analysis of MCF-10A Cells
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MCF-10A cells were cultured in 3-D using Matrigel, as previously described [11 (link), 44 (link)]. Cell cultures in chamber slides were fixed with 4% paraformaldehyde, followed by staining for immunofluorescence using anti-ezrin antibody (3C12) from Santa Cruz Biotechnology (Santa Cruz, CA), Alexa 488-conjugated anti-mouse IgG and Hoechst 33342 from Life Technologies, Grand Island, NY.
For immunofluorescence using antibodies against the EMT markers and Ki67, MCF10A cells were densely plated on poly-lysine coated coverslips. After 14-day culture in minimal medium, cells were fixed and incubated with relevant primary antibodies and secondary fluorescein-conjugated horse anti-mouse IgG antibody (#FI-2000, Vector Laboratories, Burlingame, CA) or Texas Red®-conjugated goat anti-rabbit IgG antibody (#TI-1000, Vector Laboratories). The slides were mounted with Vectashield Mounting Medium plus DAPI (#H-1200, Vector Labs)
Samples were then photographed using Nikon C2 Confocal Microscope or Zeiss Axiovert 200M Microscope. For quantification of the size of acini or “islands”, the images in10x magnification were acquired by TissueFAXS 200 (Tissuegnostics, Vienna, Austria) or by Zeiss Axiovert 200M, followed by analysis using the ImageJ software.
For immunofluorescence using antibodies against the EMT markers and Ki67, MCF10A cells were densely plated on poly-lysine coated coverslips. After 14-day culture in minimal medium, cells were fixed and incubated with relevant primary antibodies and secondary fluorescein-conjugated horse anti-mouse IgG antibody (#FI-2000, Vector Laboratories, Burlingame, CA) or Texas Red®-conjugated goat anti-rabbit IgG antibody (#TI-1000, Vector Laboratories). The slides were mounted with Vectashield Mounting Medium plus DAPI (#H-1200, Vector Labs)
Samples were then photographed using Nikon C2 Confocal Microscope or Zeiss Axiovert 200M Microscope. For quantification of the size of acini or “islands”, the images in10x magnification were acquired by TissueFAXS 200 (Tissuegnostics, Vienna, Austria) or by Zeiss Axiovert 200M, followed by analysis using the ImageJ software.
9
Hemocyte Isolation and Imaging of Drosophila Fat Body
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Larvae were bled in Schneider’s media and the hemolymph was plated on a cover-slip bottom dish for attachment for 45 minutes. Hemocytes were fixed in 4% para-formaldehyde for 20 minutes followed by wash with PBS. Cells were then permeabilized with 0.4% NP40 and blocked with 20% goat serum for 1 hour. The preps were incubated with primary antibodies at 4 °C overnight. Secondary antibody staining was performed following this and the hemocytes were incubated with DAPI to mark the nuclei. Images were taken in LSM510 Meta Confocal microscope. For hemocyte count, images were taken in Olympus IX81 microscope.
Larvae or adult flies were dissected in 1X PBS to isolate the fat body. The fat body preps were fixed in 4% para-formaldehyde for 20 minutes followed by wash with PBS, permeabilized with 0.1% Triton-X 100 and blocked with 20% normal goat serum for 1 hour. The preps were then incubated with primary antibodies at 4 °C overnight. Secondary antibody staining was then performed using Alexa-488 conjugated anti-mouse IgG and Alexa-568 tagged anti-rabbit IgG antibodies (Life Technologies). The fat body preps were mounted in DAPI. Images were taken in Zeiss LSM510 Meta and LSM880 confocal Microscope. Autofluorescence was taken care of by optimizing the confocal microscope settings using the no primary antibody control.
Larvae or adult flies were dissected in 1X PBS to isolate the fat body. The fat body preps were fixed in 4% para-formaldehyde for 20 minutes followed by wash with PBS, permeabilized with 0.1% Triton-X 100 and blocked with 20% normal goat serum for 1 hour. The preps were then incubated with primary antibodies at 4 °C overnight. Secondary antibody staining was then performed using Alexa-488 conjugated anti-mouse IgG and Alexa-568 tagged anti-rabbit IgG antibodies (Life Technologies). The fat body preps were mounted in DAPI. Images were taken in Zeiss LSM510 Meta and LSM880 confocal Microscope. Autofluorescence was taken care of by optimizing the confocal microscope settings using the no primary antibody control.
10
Immunofluorescence Assay for Tubulin and Proliferation
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Cells were fixed with ice-cold 4% PFA (20 min, RT), blocked in 5% normal goat serum/PBS (20 min) and incubated with anti-α-tubulin (1:10) and Ki67 (1:400) antibodies. Secondary antibodies were Alexa488-conjugated anti-mouse IgG and Alexa594-conjugated anti-rabbit IgG both (Life Technologies). Cell nuclei were counterstained using Prolong Gold mounting media with DAPI (Life Technologies). Images were acquired with a Zeiss Axio Scope.A1 microscope using ZEN 2 – blue edition software (Zeiss). Images were processed using Fiji.
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