Alexa546 conjugated anti mouse igg

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa546-conjugated anti-mouse IgG is a secondary antibody used in immunological assays. It is specific for mouse immunoglobulin G (IgG) and is conjugated to the fluorescent dye Alexa Fluor 546. This product can be used to detect and visualize mouse IgG in various applications, such as immunofluorescence and flow cytometry.

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Lab products found in correlation

Alexa 488 conjugated anti rabbit igg, by Thermo Fisher Scientific (5 mentions) Lsm 700, by Zeiss (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Hrp conjugated anti rabbit igg, by GE Healthcare (2 mentions) Horseradish peroxidase hrp conjugated anti mouse igg, by GE Healthcare (2 mentions) Alexa546 conjugated anti rabbit igg, by Thermo Fisher Scientific (1 mentions) Anti tom20, by Abcam (1 mentions) 710 meta, by Zeiss (1 mentions) Anti α sma, by Abcam (1 mentions) Anti vimentin, by Cell Signaling Technology (1 mentions) Incellis cell imager, by Bertin Technologies (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Dojindo Laboratories (1 mentions) Rad51, by Cell Signaling Technology (1 mentions) Anti γh2ax, by Merck Group (1 mentions) Rat anti mouse cd68 mca1957, by Bio-Rad (1 mentions) Alexa 568 conjugated anti rabbit igg, by Abcam (1 mentions) Rabbit anti iba1 antibody, by Fujifilm (1 mentions) 4 6 diamidino 2 phenylindole dihydrochloride dapi, by Thermo Fisher Scientific (1 mentions) Epifluorescence microscope, by Nikon (1 mentions) Fluorescent microscope, by Olympus (1 mentions)

Close spelling variants of this product

Alexa546-conjugated anti-mouse IgG Ab Anti-mouse-Alexa546 Mouse anti-IgG

11 protocols using alexa546 conjugated anti mouse igg

Immunofluorescence Staining of Cellular Markers

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PSCs or tumor cells were seeded in eight-well slide dishes (Ibidi, Gräfelfing, Germany) at 5 × 10⁴/well and incubated for 24 h. Then cells were fixed with 4% formaldehyde and blocked with normal horse serum for 30 min. Slides were incubated with primary antibody (anti-αSMA, 1:200, Abcam, Cambridge, UK; anti-IL-17RB, 1:200, ProteoTech, Attendorn, Germany; anti-vimentin, 1:200, Cell Signaling Technology, Danvers, MA, USA; anti-ATP5β, 1:500, Abcam, Cambridge, UK; anti-Tom20, 1:400, Abcam, Cambridge, UK) overnight at 4 °C. Subsequently, cells were incubated with Alexa 546-conjugated anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA) or Alexa 546-conjugated anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. Finally, nuclear DNA was counterstained with DAPI (4′,6-diamidino-2-phenylindole, 1 mg/mL) for 15 min. After washing with PBS, images were acquired using a fluorescence microscope (to generate Figure 1C, InCellis Cell Imager, Bertin Instruments, Montigny-le-Bretonneux, France) or a confocal microscope (to generate Figure 6A, Zeiss Meta 710, Zeiss, Jena, Germany).

Li J., Wu X., Schiffmann L., MacVicar T., Zhou C., Wang Z., Li D., Camacho O.V., Heuchel R., Odenthal M., Hillmer A., Quaas A., Zhao Y., Bruns C.J, & Popp F.C. (2021). IL-17B/RB Activation in Pancreatic Stellate Cells Promotes Pancreatic Cancer Metabolism and Growth. Cancers, 13(21), 5338.

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Visualizing DNA Damage Response Markers

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After 24 h of culture, MEF or ES cells were treated with 10 ng/ml NCS (Sigma-Aldrich) for 30 min at 37 °C just prior to fixation with 4% (w/v) PFA. Then, permeabilization was performed for 10 min with 0.2% Triton X-100, and the cells were blocked using 5% (w/v) BSA/TBS and incubated with anti-γ-H2AX (EMD Millipore, Darmstadt, Germany), Rad51 (Cell Signaling Technology, Massachusetts, USA) and 53BP1 (Novus, Missouri, USA) antibodies overnight at 4 °C. After three washes, the cells were incubated with Alexa 488-conjugated anti-rabbit IgG (Thermo Fisher Scientific) or Alexa 546-conjugated anti-mouse IgG (Thermo Fisher Scientific) for 1–2 h at room temperature. The nuclei were counterstained with 100 nM 4′,6-diamidino-2-phenylindole (DAPI) (Dojindo, Japan), images were captured using a laser scanning confocal microscope (LSM 700, Carl Zeiss, Germany).

Hirotsune S., Kiyonari H., Jin M., Kumamoto K., Yoshida K., Shinohara M., Watanabe H., Wynshaw-Boris A, & Matsuzaki F. (2020). Enhanced homologous recombination by the modulation of targeting vector ends. Scientific Reports, 10, 2518.

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Immunostaining of GD3 and GD2 in Mice

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Mouse anti-GD3 monoclonal antibody (mAb) (R24) was obtained from Dr. L. J. Old (Memorial Sloan-Kettering Cancer Center, New York, USA). Anti-GD2 mAb 220-51 was as described previously.^{31 (link)} Rat anti-mouse CD68 (MCA1957) and rabbit anti-mouse iNOS (PA1-036) were purchased from AbDSerotec (Kidlington, UK). Rabbit anti-Iba1 antibody was from Wako (Cat. No. 019-19741, Osaka, Japan). Alexa568-conjugated anti-rabbit IgG and Alexa488-conjugated anti-rat IgG were purchased from Abcam (Cambridge, UK). Alexa546-conjugated anti-mouse IgG and DAPI were from Thermo Fisher Scientific (Waltham, MA, USA).

Zhang P., Ohkawa Y., Yamamoto S., Momota H., Kato A., Kaneko K., Natsume A., Farhana Y., Ohmi Y., Okajima T., Bhuiyan R.H., Wakabayashi T., Furukawa K, & Furukawa K. (2021). St8sia1-deficiency in mice alters tumor environments of gliomas, leading to reduced disease severity. Nagoya Journal of Medical Science, 83(3), 535-549.

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Immunostaining for Myosin Heavy Chain

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Immunostaining for myosin heavy chain (MHC) was visualized in myotubes using immunofluorescence as previously described by Choi et al. [10 (link)]. Cells were plated on glass coverslips in 6-well dishes. After 24 h of PA treatment, the cells were washed with phosphate-buffed saline (PBS) and fixed in 4% paraformaldehyde in PBS. After permeabilization with 0.1% saponin, the slides were blocked with 1% BSA and incubated with an anti-MHC mouse monoclonal antibody (MF20; Developmental Studies Hybridoma Bank (DSHB), USA; 1:300 dilution) for overnight at 4°C. This was followed by incubation with Alexa-546-conjugated anti-mouse IgG (Molecular Probes, USA), and the nuclei were stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes). Images were captured using a fluorescence microscope (Olympus IX71, Japan). All cell nuclei and nuclei within myotubes were counted using the NIH Image J software (National Institute of Health, USA). The fusion index was measured as previous described [27 (link)].

Kim J.S., Lee H., Yoo A., Jeong H.Y., Jung C.H., Ahn J, & Ha T.Y. (2023). Gromwell (Lithospermum erythrorhizon) Attenuates High-Fat-Induced Skeletal Muscle Wasting by Increasing Protein Synthesis and Mitochondrial Biogenesis. Journal of Microbiology and Biotechnology, 34(3), 495-505.

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Immunostaining of Myosin Heavy Chain

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Immunostaining for myosin heavy chain was visualized in myotubes by immunofluorescence as described previously [63 (link)]. In brief, cells were plated on glass cover slips in six-well dishes. After 24 or 48 hours of H₂O₂ treatment, the cells were washed with PBS and fixed in 4% formaldehyde in PBS. MF20 (Developmental Hybridoma Bank, IA) was used to detect the cellular location of this protein on myotubes, whereas desmin was used to identify myoblasts. The primary antibodies were followed by Alexa 546 conjugated anti-mouse IgG (Molecular Probes, Eugene, OR) and nuclei were stained with (DAPI). Samples were visualized using an Epi-fluorescence microscope (Nikon, Inc., Melville, NY), and images were obtained using a SPORT RT camera (Diagnostic Instruments, Sterling Height, MI).

Haramizu S., Asano S., Butler D.C., Stanton D.A., Hajira A., Mohamed J.S, & Alway S.E. (2017). Dietary resveratrol confers apoptotic resistance to oxidative stress in myoblasts. The Journal of nutritional biochemistry, 50, 103-115.

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Quantifying DNA Damage Foci in Cells

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Cells, diluted to appropriate numbers, were grown on a glass slide after exposure to olaparib (1 µM) and/or oxaliplatin (10 µM) for 36 h. Mouse monoclonal anti-γH2AX (1:200; CST, Boston, MA, USA) antibody was used as the primary antibody. Alexa-546-conjugated anti-mouse IgG (Molecular Probes) was used for visualization of γH2AX. We then stained with 4′,6-diamidino-2-phenylindole (DAPI) (Nanjing KeyGEN Biotech. Co., Ltd., Jiangsu, People’s Republic of China). Foci were observed with an Olympus fluorescent microscope. For quantification of foci, clear and easily distinguished dots of certain brightness were counted as positive foci. The number of foci was counted in 100 cells of each sample by visual inspection, and the average number of foci per cell was calculated.

Xu K., Chen Z., Cui Y., Qin C., He Y, & Song X. (2015). Combined olaparib and oxaliplatin inhibits tumor proliferation and induces G2/M arrest and γ-H2AX foci formation in colorectal cancer. OncoTargets and therapy, 8, 3047-3054.

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Antibody and Lectin Toolkit for Cell Biology

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The following antibodies and lectins were used: anti-GnT-V (mouse, clone 706824, R&D Systems), anti-α1 sodium potassium ATPase (mouse, clone 464.6, Abcam), anti-LAMP1 (rabbit, Abcam), anti-GAPDH (mouse, clone 6C5, Merck Millipore), anti-GRP78 (BiP) (rabbit, Abcam), anti-GM130 (rabbit, clone D6B1) (Cell Signaling Technology), anti-Golgin97 (rabbit, clone D8P2K, Cell Signaling Technology), anti-Rab5 (rabbit, clone C8B1, Cell Signaling Technology), anti-Rab7 (rabbit, Cell Signaling Technology), anti-Rab11 (rabbit, Cell Signaling Technology), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (GE Healthcare), HRP-conjugated anti-rabbit IgG (GE Healthcare), unconjugated-L4-PHA (J-Chemical), biotinylated-SSA (J-Chemical), biotinylated-RCA (Vector Laboratories), FITC-conjugated L4-PHA (J-Oil Mills), Rhodamine-conjugated L4-PHA (Vector Laboratories), Alexa546-conjugated anti-mouse IgG (Invitrogen), Alexa488-conjugated anti-rabbit IgG (Invitrogen). L4-PHA was conjugated to HRP using a Peroxidase Labeling Kit-NH2 (Dojindo) as described previously (22 (link)).

Osuka R.F., Hirata T., Nagae M., Nakano M., Shibata H., Okamoto R, & Kizuka Y. (2022). N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates. The Journal of Biological Chemistry, 298(3), 101666.

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Detect Surface and Intracellular KIR2DL5

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Forty-eight hours after transfection, HEK-293T cells were stained with anti-FLAG M2 mAb, fixed in PBS with 4% paraformaldehyde, and incubated with Alexa546-conjugated anti-mouse IgG (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) to detect KIR2DL5–FLAG molecules on the surface. Then, cells were treated with 0.3% Triton X-100, and re-incubated with anti-FLAG M2 mAb, followed by Alexa448-labeled anti-mouse IgG (Invitrogen), to detect intracellular KIR2DL5 molecules. After this staining strategy, based on Ref. (21 (link)), cells were visualized on poly-l-lysine-coated glass-bottom dishes on a confocal laser-scanning microscope (TCS SP5, Leica Microsystems CMS GmbH, Mannheim, Germany) using Argon (488 nm) and Helium-Neon (543 nm) lasers and a 20×/0.5 lens. Images were acquired using the LAS AF SP5 software (Leica).

Cisneros E., Estefanía E, & Vilches C. (2017). Allelic Polymorphism Determines Surface Expression or Intracellular Retention of the Human NK Cell Receptor KIR2DL5A (CD158f). Frontiers in Immunology, 7, 698.

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Immunohistochemistry Analysis of Adult and Infant Macula

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Immunohistochemistry experiments were performed on frozen human tissue sections fixed in 4% paraformaldehyde [35 (link)]. Infant (n=3) and aged adult (n=5) maculas were evaluated. Sections were blocked with 1 mg/mL of bovine serum albumin for fifteen minutes and were subsequently incubated with 50 μL of 1X TrueBlack autofluorescence quencher (Biotinium, Hayward CA) for 60 seconds followed by three five-minute washes, according to the manufacturer’s instructions. This treatment quenches the very abundant autofluorescence present in adult RPE cells. Sections were incubated with anti-CD34 (1:100, Abcam, EP373Y) and anti-ICAM1 (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA, P2A4) primary antibodies for one hour. Negative controls were additionally obtained by omitting each primary antibody. After washing, Alexa-546-conjugated anti-mouse IgG (1:200, Invitrogen) and Alexa-488-conjugated anti-rabbit IgG (1:200, Invitrogen) secondary antibodies resuspended in PBS supplemented with 100 μg/mL diamidino-phenyl-indole (DAPI, Sigma) were added to each section for 30 minutes. Sections were washed and coverslipped, and photographs were acquired with a confocal microscope (Leica DM 2500 SPE). In all steps, from immunofluorescent labeling to finalization of figures, all infant and adult samples were treated identically.

Voigt A.P., Whitmore S.S., Mulfaul K., Chirco K.R., Giacalone J.C., Flamme-Wiese M.J., Stockman A., Stone E.M., Tucker B.A., Scheetz T.E, & Mullins R.F. (2020). Bulk and single-cell gene expression analyses reveal aging human choriocapillaris has pro-inflammatory phenotype. Microvascular research, 131, 104031.

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Visualizing IRAK and F-actin

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Anti-IRAK (Cell Signaling) and anti-F-actin (Abcam, Cambridge, MA, USA) primary antibodies and Alexa 488-conjugated anti-rabbit-IgG and Alexa 546-conjugated anti-mouse-IgG secondary antibodies (all from Invitrogen, Carlsbad, CA, USA) were used. Image acquisition and processing were performed using confocal fluorescence microscopes and the FV10-ASW 2.0 Viewer (Olympus, Center Valley, PA, USA).

Oh K., Lee O.Y., Park Y., Seo M.W, & Lee D.S. (2016). IL-1β induces IL-6 production and increases invasiveness and estrogen-independent growth in a TG2-dependent manner in human breast cancer cells. BMC Cancer, 16(1), 724.

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