Alexafluor568 goat anti mouse igg1

Manufactured by Thermo Fisher Scientific

Sourced in United States

AlexaFluor568 goat anti-mouse IgG1 is a fluorescently labeled secondary antibody used for detection and visualization in various immunoassays and imaging techniques. It binds specifically to the IgG1 subclass of mouse immunoglobulins and is conjugated with the AlexaFluor568 fluorescent dye, which has an excitation/emission spectrum optimized for red fluorescence detection.

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Lab products found in correlation

Dylight 488 goat anti rabbit igg, by Thermo Fisher Scientific (3 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (3 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Am tirf mc system, by Leica (2 mentions) Monoclonal mouse anti ctbp2 antibody, by BD (2 mentions) Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions) Normal goat serum (ngs), by Cell Signaling Technology (2 mentions) Pbs 0.3 triton x 100, by Merck Group (2 mentions) Monoclonal mouse anti glur2 antibody, by Merck Group (2 mentions) Goat anti mouse igg2 alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Acridine orange, by Merck Group (1 mentions) Hyclone dmem without phenol red, by GE Healthcare (1 mentions) Tmr phalloidin, by Merck Group (1 mentions) Arpc3, by Merck Group (1 mentions) Wave2, by Cell Signaling Technology (1 mentions) Total akt, by Cell Signaling Technology (1 mentions) Plc γ py783, by Cell Signaling Technology (1 mentions) Alexa fluor 488 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Mouse IgG1 (32 citations) Goat anti-mouse IgG1 (22 citations)

14 protocols using alexafluor568 goat anti mouse igg1

Detailed Cell Culture Reagents and Methods

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Dulbecco’s Modified
Eagle’s Medium (DMEM), phosphate buffered saline (PBS; pH 7.4),
0.25% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), fetal
bovine serum (FBS), penicillin–streptomycin (Penstrep), and
Hank’s Balanced Salt Solution (HBSS) were obtained from Gibco,
Life Technologies Corporation (Painsley, United Kingdom) and used
for cell culture experiments. MEM Nonessential Amino Acid Solution
(100×; NEAA), Bovine Serum Albumin (BSA), and Acridine Orange
(AO) were purchased from Sigma-Aldrich (Munich, Germany). HyClone
(DMEM without phenol red) was obtained from GE Healthcare Life Sciences
(Logan, UT, USA). Alexa Fluor 488 Phalloidin, Hoechst 33342, and primary
occludin monoclonal antibody were obtained from Thermo Fisher Scientific
(Vienna, Austria). Alexa Fluor 568 goat anti-mouse IgG1 was purchased
from Thermo Fisher Scientific (Waltham, MA, USA).

Zeiringer S., Wiltschko L., Glader C., Reiser M., Absenger-Novak M., Fröhlich E, & Roblegg E. (2023). Development and Characterization of an In Vitro Intestinal Model Including Extracellular Matrix and Macrovascular Endothelium. Molecular Pharmaceutics, 20(10), 5173-5184.

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Modulation of T Cell Actin Dynamics

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Activated T cells were stimulated in the presence of ethanol or GML with plate-bound anti-CD3 on glass coverslips for 15 min. In experiments with inhibitors of cytoskeletal processes, 100 μM Colchine (Cayman Chemicals), 10 μM SMIFH2 (Calbiochem), or 10 μM CK-666 (Calbiochem) solubilized in DMSO were added to the activated T cells at the same time as the GML or solvent. Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.25% Triton-X for 5 min, and then stained with the appropriate reagents. To detect actin, TMR-Phalloidin (Sigma) was incubated with the fixed and permeabilized cells for 2 hours at 37°C. To detect microcluster formation of signaling proteins, cells were stained with antibodies specific for Arp2 (Cell Signaling), WAVE2 (Cell Signaling), phosphorylated WASp Y290 (Invitrogen), ARPC3 (Millipore), phosphorylated SLP-76 Y128 (BD Pharmingen), and phosphorylated LAT Y191 (Millipore). Staining was detected with the conjugated secondary antibodies DyLight 488 Goat anti-rabbit IgG or Alexa Fluor 568 goat anti-mouse IgG1 (Thermo Fisher). TIRF images were captured by the Leica AM TIRF MC system using a 100x magnification oil immersion objective lens at the University of Iowa Central Microscopy Research Facility. Actin ring structures were imaged using the same microscope using the epifluorescence channels.

Zhang M.S., Tran P.M., Wolff A.J., Tremblay M.M., Fosdick M.G, & Houtman J.C. (2018). Glycerol Monolaurate (GML) induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters. Science signaling, 11(528), eaam9095.

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Imaging TCR Signaling Protein Microclusters

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APBTs were stimulated in the presence of 0.1% ethanol control or 10 μg/ml GML with plate-bound anti-CD3 on glass coverslips, fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton-X, and stained as described below. To detect microcluster formation of TCR signaling proteins, antibodies specific for LAT pY226 (BD Pharmingen), PLC-γ pY783 (Cell Signaling, total P85 PI3K (Millipore), and total AKT (Cell signaling) were incubated with fixed and permeabilized cells overnight for 4 °C. Subsequently, conjugated secondary antibodies DyLight 488 Goat anti-rabbit IgG and Alexa Fluor 568 goat anti-mouse IgG1 (Thermo Fisher) were incubated with the cells at room temperature for 2 hours. All images were captured by the Leica AM TIRF MC system using 100x magnification oil immersion objective lens at room temperature at the University of Iowa Central Microscopy Research Facility. Phospho-specific LAT pY226 antibody was used due to its ability to robustly image LAT micrcoclusters in TIRF17 (link).

Zhang M.S., Sandouk A, & Houtman J.C. (2016). Glycerol Monolaurate (GML) inhibits human T cell signaling and function by disrupting lipid dynamics. Scientific Reports, 6, 30225.

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Immunofluorescence Staining of EC and SMC

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Passage 1 EC and SMC were grown to confluence on plastic dishes or on chitosan/PCL membranes. After that, they were fixed with 4% PFA (paraformaldehyde) for 10 min, permeabilized with 0.05% Triton X-100 for 10 min, and blocked with 1% BSA (bovine serum albumin). The cells were stained with primary antibodies overnight at 4 °C, washed with PBS and incubated with secondary antibodies for 1 h at room temperature. The stained cells were analysed with an inverted fluorescence microscope (Nikon Ti-E) using Nikon AR software.
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).

Zakharova I.S., Zhiven’ M.K., Saaya S.B., Shevchenko A.I., Smirnova A.M., Strunov A., Karpenko A.A., Pokushalov E.A., Ivanova L.N., Makarevich P.I., Parfyonova Y.V., Aboian E, & Zakian S.M. (2017). Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts. Journal of Translational Medicine, 15, 54.

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Immunofluorescence Analysis of VACV Infection

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Fluorescent microscopy for immunofluorescence was carried out using a Zeiss Axiostar plus epifluorescent microscope and images were captured with an Optronics camera system. Cells were grown on sterile glass coverslips, infected (MOI = 5) with ECTV, and fixed at the indicated time points with 10% formalin for 10 min at room temperature. For virus factory analysis, cells were then immediately mounted on a glass slide with ProLong Gold antifade reagent with 4’-6-diamidino-2-phenylindole (DAPI; Life Technologies) and allowed to cure overnight prior to visualization. For surface B5 staining, unpermeabilized cells were incubated—after fixation—in blocking buffer [1% bovine serum albumin (BSA; Gemini BioProducts) and 2% FBS (Gemini BioProducts) in 1x PBS] for 20 min and then incubated for 40 min with anti-B5 monoclonal antibody (BEI resources NR-553) diluted in blocking buffer. After three washes with 1x PBS, Alexa Fluor 568 goat anti-Mouse IgG1 (Life Technologies) diluted in blocking buffer was applied to cells for 20 min. Finally, the coverslips were mounted as described above on a glass slide with ProLong Gold antifade reagent with DAPI. A Leica DM-IL LED inverted epifluorescent microscope was used to visualize fluorescence from VACVΔE3LΔK3L infected cells. These images were then processed using QImaging software.

Hand E.S., Haller S.L., Peng C., Rothenburg S, & Hersperger A.R. (2015). Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells. PLoS ONE, 10(3), e0119189.

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Immunostaining of Mouse Cochlear Tissues

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Immunostaining was performed as previously described with minor modifications.^{7 (link),9} Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, MA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4°C overnight. After adequate decalcification by incubation in 500mM EDTA (3–6 days), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories.¹² The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, MO) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technologies, MA). Pre-synaptic ribbons and post-synaptic densities were labelled using a monoclonal mouse anti-CtBP2 antibody (1:200, BD Biosciences, CA) and a monoclonal mouse anti-GluR2 antibody (1:2000, Sigma-Aldrich, MO), respectively. Following this, the tissue was incubated with the corresponding secondary antibodies, goat anti-mouse IgG2 Alexa Fluor^® 488 and goat anti-mouse IgG1 Alexa Fluor^® 568 (1:1000, ThermoFisher Scientific, MA). Nuclei were counterstained with DAPI. The labelled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific, MA).

Kennedy C.L., Shuster B., Amanipour R., Milon B., Patel P., Elkon R, & Hertzano R. (2023). Metformin Protects Against Noise-Induced Hearing Loss in Male Mice. Otology & Neurotology, 44(9), 956-963.

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Immunofluorescent Analysis of Stem Cell Markers

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Immunofluorescent staining was employed to assess the presence of OCT4 and p21. Initially, PDLSCs were cultured in 8-multiwell chambers from Thermo Fisher Scientific (Carlsbad, CA, USA). Following a 24-h incubation at 37 °C to facilitate cellular adhesion, the cells were fixed using 4% paraformaldehyde, permeabilized using TBS containing 0.5% Triton, and then blocked with TBS containing 3% bovine serum albumin (BSA; Sigma-Aldrich). The subsequent steps involved incubating the cells with primary antibodies; p21 was incubated for 1 h at room temperature, while OCT4 required 3 h. This was followed by an incubation with secondary antibodies conjugated to a fluorophore for 1 h at room temperature, as reported elsewhere [23 (link)] Unconjugated monoclonal antibodies directed to human OCT4 (9B7), p21 (R.229.6) and their respective secondary antibodies goat anti-Mouse IgG1 Alexa Fluor 568 and goat anti-rabbit IgG DyLight 488 were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Nuclei were stained through SlowFade Gold antifade reagent with DAPI by Invitrogen (Thermo Fisher Scientific). Analysis was performed through a Leica SP8 inverted confocal microscope (Leica, Wetzlar, Germany).

Roato I., Baima G., Orrico C., Mosca Balma A., Alotto D., Romano F., Ferracini R., Aimetti M, & Mussano F. (2023). Senescent Markers Expressed by Periodontal Ligament-Derived Stem Cells (PDLSCs) Harvested from Patients with Periodontitis Can Be Rejuvenated by RG108. Biomedicines, 11(9), 2535.

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Immunostaining of Cochlear Synapses

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Immunostaining was performed as previously described [16 (link)] with minor modifications. Briefly, tissue was fixed by transcardial perfusion of 4% paraformaldehyde (PFA, Alfa Aesar, Haverhill, MA, USA), followed by harvest of the temporal bones and further fixation in 4% PFA at 4 °C overnight. After adequate decalcification by incubation in 500 mM EDTA (3–6 days), the cochlear ducts were dissected as described by the Eaton-Peabody Laboratories [87 ]. The tissue was permeabilized with PBS-0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and blocked in permeabilization buffer supplemented with 5% normal goat serum (Cell Signaling Technology, Danvers, MA, USA). Pre-synaptic ribbons and post-synaptic densities were labelled using a monoclonal mouse anti-CtBP2 antibody (1:200, BD Biosciences, San Jose, CA, USA) and a monoclonal mouse anti-GluR2 antibody (1:2000, Sigma-Aldrich, St. Louis, MO, USA), respectively, followed by incubation with the corresponding secondary antibodies, goat anti-mouse IgG2 Alexa Fluor^® 488 and goat anti-mouse IgG1 Alexa Fluor^® 568 (1:1000, ThermoFisher Scientific, Waltham, MA, USA). Nuclei were counterstained with DAPI. The labelled tissue was mounted with the ProLong Gold antifade reagent (ThermoFisher Scientific, Waltham, MA, USA).

Shuster B., Casserly R., Lipford E., Olszewski R., Milon B., Viechweg S., Davidson K., Enoch J., McMurray M., Rutherford M.A., Ohlemiller K.K., Hoa M., Depireux D.A., Mong J.A, & Hertzano R. (2021). Estradiol Protects against Noise-Induced Hearing Loss and Modulates Auditory Physiology in Female Mice. International Journal of Molecular Sciences, 22(22), 12208.

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Cardiomyocyte Immunohistochemistry and Analysis

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Hearts were cryogenically preserved and cryosectioned according to standard procedures detailed in Supplementary Methods. Sections were mounted on glass slides and IHC was performed using 1:1,000 anti-α-actinin mouse monocolonal antibody (Sigma, A7811) to label cardiomyocytes, followed by AlexaFluor 568 goat anti-mouse IgG₁ (Molecular Probes) at 1:250. Following IHC, sections were incubated for 10 min with 5 μg/ml wheat germ agglutinin (WGA)—Alexa 647 [Molecular Probes; for the determination of cross-sectional area (CSA)] in 1X PBS.

Farrell E.T., Grimes A.C., de Lange W.J., Armstrong A.E, & Ralphe J.C. (2017). Increased Postnatal Cardiac Hyperplasia Precedes Cardiomyocyte Hypertrophy in a Model of Hypertrophic Cardiomyopathy. Frontiers in Physiology, 8, 414.

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Immunohistochemistry Staining of Cardiac Tissue

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IHC on paraffin-embedded hearts were performed according to standard methods as detailed in Supplementary Methods, using the following antibodies: 1:20 anti-α-tropomyosin (CH-1; Developmental Studies Hybridoma Bank, University of Iowa, IA) to label myocardium, and 1:200 anti-Ki67 rabbit polyclonal antibody (Abcam, ab15580) to label cells actively participating in the cell cycle (Figure 2). For secondary detection, sections were incubated in 1:200 AlexaFluor 568 goat anti-mouse IgG₁ and Alexafluor 488 goat anti-rabbit IgG_(H+L)secondary antibodies (Molecular Probes, Eugene, OR, USA). In each case, as a control, similar sections were incubated with secondary antibodies only. Sections were coverslipped using warmed ProLong Gold Antifade Reagent (Invitrogen) with 4′,6-diamidino-2-phenylindole (DAPI) to label nuclei.

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