Alexa fluor 546 goat anti mouse igg1

Manufactured by Thermo Fisher Scientific

Sourced in Japan, United States

Alexa Fluor 546 goat anti-mouse IgG1 is a secondary antibody conjugated with Alexa Fluor 546 dye. It is designed to detect and bind to mouse IgG1 primary antibodies in immunological applications.

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Goat anti-mouse IgG1 Alexa 546 Goat anti-mouse Alexa Fluor 546 Anti-mouse Alexa Fluor 546 Goat anti-mouse Alexa 546 Alexa 546 anti-goat Alexa Fluor 546 Goat anti-mouse IgG1 Anti-mouse Alexa Fluor Alexa 546 Mouse IgG1

8 protocols using alexa fluor 546 goat anti mouse igg1

Immunofluorescence Antibody Labeling Protocol

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Alexa Fluor 546 goat anti-mouse IgG1 (A21123), Alexa Fluor 546 goat anti-mouse IgG2a (A21133), Alexa Fluor 488 donkey anti-rabbit (A21206), Alexa Fluor 405 goat anti-rabbit (A31556), Alexa Fluor 488 goat anti-mouse IgM (A21042), and Alexa Fluor 647 goat anti-mouse IgG (A21235) were obtained from Life Technologies (Table 1).

Riquelme D., Silva I., Philp A.M., Huidobro-Toro J.P., Cerda O., Trimmer J.S, & Leiva-Salcedo E. (2018). Subcellular Localization and Activity of TRPM4 in Medial Prefrontal Cortex Layer 2/3. Frontiers in Cellular Neuroscience, 12, 12.

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Internalization of Extracellular Vesicles in Rat Embryo Fibroblasts

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REFs were prepared from Wt rats at E14.5. Embryos were dissociated by trypsin treatment (Sigma-Aldrich), and the single cells were cultured in DMEM including 10% FBS and antibiotic-antimycotic solution at 37 °C in 5% CO₂. Serum-derived EVs from Wt or Tg rats were isolated using ultracentrifugation. The EVs were visualized using a PKH67 fluorescence labelling kit (Sigma-Aldrich). Serum EVs were incubated with REFs on 24-well glass bottom plates for 10–11 hours at 37 °C in 5% CO₂. After being washed, the cultured cells were fixed with 4% PFA. The EVs from Tg rats were detected using anti-human CD63 antibody after having been transferred into REFs. To analyse localization of EVs in REFs, anti-LAMP1 antibody (1:250, Sigma-Aldrich) was used. After the cells were stained with Alexa Fluor 546 Goat anti-Mouse IgG₁ (1:2000; Life Technologies), Alexa Fluor 546 Goat anti-Rabbit IgG (1:2000; Life Technologies) and Alexa Fluor 488 Goat anti-Rabbit IgG (1:200; Life Technologies) antybodies, internalization of EVs were captured with a FV1000 confocal microscope (OLYMPUS, Tokyo, Japan) and a BIOREVO BZ-9000 fluorescence microscopy (Keyence, Osaka, Japan). Hoechst 33342 was used to stain cell nuclei.
All experimental protocols were approved by the National Institute of Neuroscience, National Center of Neurology and Psychiatry, and National Cancer Centre Research Institute, Japan.

Yoshimura A., Kawamata M., Yoshioka Y., Katsuda T., Kikuchi H., Nagai Y., Adachi N., Numakawa T., Kunugi H., Ochiya T, & Tamai Y. (2016). Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids. Scientific Reports, 6, 31172.

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Multicolor Immunofluorescence Labeling

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The following reagents were used: 100 µg/ml HEL (Sigma), 100 µg/ml BSA (Euromedex), 40% polyacrylamide (PAA, Biorad), 2% bis-poylacrylamide (Biorad), 3-aminopropyltrimethoxysilane (Sigma), 0.2 µm Alexa647 Fluospheres (Thermo Fisher, F8807), Sigmacote (Sigma), ammonium persulfate (Sigma), TEMED (ICN Biomedicals), Sulfo-SANPAH (Thermo Fisher), and the Alexa555 protein labeling kit (A30007, Molecular Probes). The following primary antibodies were used: rabbit anti-HEL (Abcam,1/100), human anti-GFP (Institut Curie, 1/200), rabbit Anti-phospho-Cortactin (pTyr⁴⁶⁶) (SAB4504373, Sigma-Aldrich, 1:200), rabbit anti Clathrin (Cell Signalling 4796, 1:50), Alexa Fluor 488 Phalloidin (A12379 Invitrogen 1:200), Alexa Fluor 647-conjugated anti-phalloidin (Thermo Fisher, 1/200), anti-myosin IIA heavy chain (Covance, 1/500). The following secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies, 1/200), Alexa Fluor 488-conjugated goat anti-human IgG (Life Technologies,1/200), Alexa Fluor 405 Goat anti-Rabbit IgG (H + L) (A31556 Thermofisher 1:200), Alexa Fluor 546 Goat anti-Mouse IgG1 (A21123 Thermofisher 1:200).

Kumari A., Pineau J., Sáez P.J., Maurin M., Lankar D., San Roman M., Hennig K., Boura V.F., Voituriez R., Karlsson M.C., Balland M., Lennon Dumenil A.M, & Pierobon P. (2019). Actomyosin-driven force patterning controls endocytosis at the immune synapse. Nature Communications, 10, 2870.

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Characterizing Myogenic Progenitor Cells

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Phase-contrast microscopy, immunofluorescence staining, and qPCR analysis were performed as in [12 (link)]. Cell counting for Hoechst (Sigma–Aldrich, St. Louis, USA, B-2883) and T (Abcam, Toronto, Canada, AB20680) nuclear staining was performed as in [129 (link)]. Primary antibodies against PAX7 (Developmental Studies Hybridoma Bank, Iowa City, USA, AB528428), MKI67 (KI67)(Abcam, AB15580), MYOD1 (Abcam, AB133627), and secondary Cy3 goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, USA, 111-165-003), AlexaFluor546 goat anti-mouse IgG1 (ThermoScientific, Waltham, USA, A21123), and AlexaFluor647 goat anti-rabbit IgG (ThermoScientific, A21244) antibodies were also used. The antibody staining for PAX7, MYOD1, and KI67 were conducted similar to [13 (link)], with the exceptions that permeabilization was carried out with 0.5% TritionX-100 and 100 mM glycine in Tris-buffered saline (TBS), blocking solution was 2% bovine serum albumin (BSA), 5% goat serum, and 0.1% Tween20 in TBS, and the stains were mounted with PermaFluor (ThermoScientific, TA-006-FM). CellProfiler 3.0 was used to analyze the PAX7, MYOD1, and KI67 staining.

Shelton M., Ritso M., Liu J., O’Neil D., Kocharyan A., Rudnicki M.A., Stanford W.L., Skerjanc I.S, & Blais A. (2019). Gene expression profiling of skeletal myogenesis in human embryonic stem cells reveals a potential cascade of transcription factors regulating stages of myogenesis, including quiescent/activated satellite cell-like gene expression. PLoS ONE, 14(9), e0222946.

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Antibody staining for chicken macrophages

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Primary antibodies were as follows: mouse monoclonal IgG1 anti-chicken monocyte/macrophage [KUL01, 1:500 dilution, Cat. No. SC52603, Santa Cruz Biotechnology, Santa Cruz, CA, validated by Mast et al. (21 (link))] and mouse monoclonal IgG2a anti-BrdU (1:500 dilution, Cat. No. MA3-071, Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA). Secondary antibodies (Invitrogen; 1:1000 dilution) were: Alexa-Fluor 546 goat anti-mouse IgG1 (Cat. No. A21123) and Alexa-Fluor 633 goat anti-mouse IgG2a (Cat. No. A21136).

Keel A.J., Calderon A.J., Tejeda O.J., Starkey J.D, & Starkey C.W. (2022). Dietary Protein Source and Litter Condition Alter Broiler Chicken Intestinal Macrophage and Mitotically Active Cell Populations. Frontiers in Veterinary Science, 9, 894587.

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Quantifying Extracellular Matrix Proteins

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hPASMC were seeded on 24-well plates containing gels with stiffness of 0.4 and 25.6 kPa and treated with iloprost (10 μmol/l) or vehicle (DMSO). After 48 hours, cells were fixed with 4% formalin, blocked with 5% goat serum, and stained with mouse monoclonal anti-Pro-Collagen I (clone PCIDG10, catalog MAB1913, Millipore) and mouse monoclonal anti-Fibronectin (clone IST-9, Abcam) antibodies. After washing, cells were incubated with Alexa Fluor 546 goat anti–mouse IgG1 (Invitrogen). F-actin and nuclei were stained with Alexa Fluor 488–phalloidin and Hoechst 33342 (Invitrogen), respectively. Fluorescent images were obtained with a Nikon TE300 fluorescent microscope. The final image contrast was adjusted with MetaMorph 6.1 (Universal Imaging) by setting the background to the fluorescence intensity level of the samples in the corresponding secondary antibody–only control images. Procollagen I staining was quantified with MetaMorph 6.1 by counting the procollagen I staining–positive cells and normalizing to the total cell number in each image. Fibronectin staining was quantified with MetaMorph 6.1 by measuring the average cellular fibronectin fluorescence intensity (normalized to stained areas) and then normalized to the corresponding total cell number.

Liu F., Haeger C.M., Dieffenbach P.B., Sicard D., Chrobak I., Coronata A.M., Velandia M.M., Vitali S., Colas R.A., Norris P.C., Marinković A., Liu X., Ma J., Rose C.D., Lee S.J., Comhair S.A., Erzurum S.C., McDonald J.D., Serhan C.N., Walsh S.R., Tschumperlin D.J, & Fredenburgh L.E. (2016). Distal vessel stiffening is an early and pivotal mechanobiological regulator of vascular remodeling and pulmonary hypertension. JCI Insight, 1(8), e86987.

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Immunofluorescence Staining of CXCL12 and PDGFRα

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Paraffin sections were deparaffinized and autoclaved in citric acid buffer (pH 6.0) to facilitate heat mediated antigen retrieval. The sections were then incubated in blocking solution containing 10% goat serum (Sigma, G9023) and 10% rabbit serum (Sigma, R9023) for 1 h and then overnight with the following primary antibodies: anti-h/m anti-C-X-C motif ligand 12 (CXCL12)/Stromal cell-derived factor-1 (SDF-1), mouse monoclonal IgG1 (R&D Systems, MAB350) and rat anti-mouse anti-platelet-derived growth factor receptor alpha (PDGFRα) (BD Pharmingen, 558774) in cold place. After washing with PBS, the sections were then incubated for 2 h with the following secondary antibodies: Alexa Fluor 546 goat anti-mouse IgG₁ (Invitrogen, A21123) and Alexa Fluor 488 rabbit anti-rat IgG (Life technologies, A21210). Sections were treated with Hoechst (Dojindo, H342) for nuclear staining. Followed by washing with PBS, the samples were mounted with ProLong Gold (Invitrogen, A11008), and observed by fluorescence microscope (Leica Microsystems, LAS AF with DMI 6000B).

Sayo K., Aoki S, & Kojima N. (2016). Fabrication of bone marrow-like tissue in vitro from dispersed-state bone marrow cells. Regenerative Therapy, 3, 32-37.

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Flow Cytometry Analysis of NKT Cells

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SupT1 cells and NKTs were prepared according to the manufacturer’s instructions (Abcam immunofluorescence protocol). Briefly, cells were fixed with cytofix buffer (BD Biosciences) and permeabilized with 0.1% Triron X in PBS. Cells were then incubated with anti-Vα24Jα18 (clone 6B11, Novus biologicals) diluted at 1:800 in PBS/1% BSA. The Alexa Fluor 546 goat anti-mouse IgG1 (Invitrogen) diluted at 1:500 in PBS/1% BSA was used as a secondary Ab. Cells were mounted cover slips with one drop of ProLong Diamond Antifade Mountant with DAPI (Invitrogen). Data acquisition was performed on LSM700 Zeiss confocal microscopy using ZEN software (ZEISS Microscopy). Data analysis was performed with Fiji software.

Landoni E., Smith C.C., Fucá G., Chen Y., Sun C., Vincent B.G., Metelitsa L.S., Dotti G, & Savoldo B. (2019). A High Avidity T-Cell Receptor Redirects Natural Killer T-Cell Specificity and Outcompetes the Endogenous Invariant T-Cell Receptor. Cancer immunology research, 8(1), 57-69.

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