Alexa fluor 488 goat anti mouse

Manufactured by Thermo Fisher Scientific

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Alexa Fluor 488 goat anti-mouse is a fluorescently labeled secondary antibody used in various immunodetection techniques. It is designed to bind to primary antibodies raised in mouse, allowing for visualization and detection of target proteins or cells.

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726 protocols using alexa fluor 488 goat anti mouse

Visualizing ACSL4 and SARS-CoV-2 Infection

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ACSL4^+/+ or ACSL4^−/− cells were infected with CV-A6 at an MOI of 20. After 7 h postinfection, cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. Cells were permeabilized and immunostained with anti-dsRNA, anti-ACSL4, and anti-CNX antibodies and then stained with Alexa Fluor 488 goat anti-mouse (A11008; Invitrogen), Alexa Fluor 594 goat anti-rabbit (A11012; Invitrogen), or Alexa Fluor 647 donkey anti-goat (A21447; Invitrogen) secondary antibodies. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were examined under a confocal laser-scanning microscope (LSM780; Zeiss, Oberkochen, Germany).
LLC-MK2 cells were infected with SARS-CoV-2 at an MOI of 0.01. At 4 days postinfection, cells were washed with PBS and fixed with 4% formaldehyde for 10 min at room temperature. Cells were permeabilized and immunostained with anti-SARS-CoV-2 spike and anti-SARS-CoV-2 nucleocapsid antibodies and then stained with Alexa Fluor 488 goat anti-mouse (A11001; Invitrogen) or Alexa Fluor 594 goat anti-rabbit (A11012; Invitrogen) secondary antibodies. The nuclei were stained with DAPI. The cells were examined under a fluorescence microscope (Olympus IX71; Tokyo, Japan).

Kung Y.A., Chiang H.J., Li M.L., Gong Y.N., Chiu H.P., Hung C.T., Huang P.N., Huang S.Y., Wang P.Y., Hsu T.A., Brewer G, & Shih S.R. (2022). Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis. mBio, 13(1), e02717-21.

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Immunofluorescence Staining Protocol

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Antibody stainings were performed as described previously (Fadeev et al., 2015 (link)). anti-Tjp1aC was used 1:100, as secondary antibody Alexa Fluor 488 goat anti-mouse (Invitrogen/Molecular Probes, A11008) was used 1:400.

Mahalwar P., Singh A.P., Fadeev A., Nüsslein-Volhard C, & Irion U. (2016). Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish. Biology Open, 5(11), 1680-1690.

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Immunofluorescence Microscopy of Cells

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Cells were seeded in 12-well plates at a density of 1 × 10⁵ cells per well on coverslips overnight. Cells were treated as described above and were fixed in ice-cold methanol and permeabilized for 10 min with 0.5% Triton X-100/PBS, washed with PBS, and then blocked with 5% BSA/PBS. Cells were incubated overnight with primary antibodies diluted in 4% BSA and 0.1% Tween 20 in PBS. Cells were then washed with PBS and incubated with fluorescent secondary antibodies Alexa Fluor 488 goat anti-mouse and Alexa Fluor 555 goat anti-rabbit antibodies were from Molecular Probes (Invitrogen, Waltham, MA, USA). Negative controls were carried out using PBS instead of primary antibodies. Cells were washed, mounted, and imaged by confocal laser scanning microscopy, which was performed with an Olympus Fluo view FV 1000 microscope, using an Olympus 60x/1.20 NA UPLAN APO water immersion objective.

Leonardi D.B., Anselmino N., Brandani J.N., Jaworski F.M., Páez A.V., Mazaira G., Meiss R.P., Nuñez M., Nemirovsky S.I., Giudice J., Galigniana M., Pecci A., Gueron G., Vazquez E, & Cotignola J. (2019). Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer. International Journal of Molecular Sciences, 20(5), 1006.

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Immunofluorescence Imaging of CHES1 Protein

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For fluorescence microscopy, 4 × 10⁴ cells were plated on coverslips in six-well plates. At 24 h after plating, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then the cells were washed in 1× PBS and permeabilized using ice-cold 0.2% Triton X-100 in PBS/BSA 3% solution for 5 min. Then cells were washed three times with PBS/BSA and incubated for 1 h at room temperature with anti-CHES1 CHESC9H4 mouse monoclonal antibody (1:200; Abcam). The cells were washed and incubated for 1 h with the appropriate conjugated secondary antibody (1:4000, Alexa Fluor 488 goat anti-mouse; Molecular Probes-Invitrogen). Finally, the cells were washed three times with 1× PBS, incubated in 300 nM 4′,6-diamidino-2-phenylindole for 10 min and mounted on microscope slides. Images were obtained using a Nikon Eclipse TE2000-U microscope and MetaMorph software (Universal Imaging).

Huot G., Vernier M., Bourdeau V., Doucet L., Saint-Germain E., Gaumont-Leclerc M.F., Moro A, & Ferbeyre G. (2014). CHES1/FOXN3 regulates cell proliferation by repressing PIM2 and protein biosynthesis. Molecular Biology of the Cell, 25(5), 554-565.

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Immunofluorescence Staining of Cellular Structures

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Filters were washed in warm phosphate-buffered saline (PBS) + MgCl₂ + CaCl₂ (Sigma; D8662) and fixed in warm 4% paraformaldehyde and 100 mM sodium cacodylate at ambient temperature. After two washes in PBS, the filters were quenched (PBS, 20 mM glycine, and 75 mM ammonium chloride) for 5 min and permeabilized for 10 min in quench solution containing 0.1% Triton X-100. After being washed with PBS, the filters were blocked with PBS, 1% BSA, and 0.1% saponin, and incubated for 1 h with primary antibody diluted in PBS, 0.5% BSA, and 0.025% saponin (wash buffer). The filters were washed three times, incubated for 30 min with secondary antibody diluted in wash buffer, washed three times, and mounted onto glass slides with ProLong Gold antifade reagent (Molecular Probes, P36935). Antibodies and stains: mouse α-acetylated tubulin 1:500 (Sigma-Aldrich; T7451); rhodamine phalloidin 1:140 (Cytoskeleton; PHDR1); rabbit anti–megalin 1:1000 (MC-220, gift from Dan Biemesderfer, Yale University [ Zou et al., 2004 (link)]); Alexa Fluor 488 goat anti-mouse 1:500 (Molecular Probes; A11029); and Alexa Fluor 488 goat anti-rabbit 1:500 (Molecular Probes; A11034). Filters were imaged on a Leica TCA SP5 confocal microscope using the Plan-Apo 40× objective.

Long K.R., Shipman K.E., Rbaibi Y., Menshikova E.V., Ritov V.B., Eshbach M.L., Jiang Y., Jackson E.K., Baty C.J, & Weisz O.A. (2017). Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway. Molecular Biology of the Cell, 28(19), 2508-2517.

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Evaluating Myogenic Purity and Morphology

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By using a mouse monoclonal anti-Desmin antibody (D33; Dako, Produktionvej, Denmark), the myogenic purity and cellular morphology were determined. Desmin is a myogenic lineage specific protein that will not be expressed in fibroblasts. The cells were first fixed in cold ethanol for 5 min. After the blocking step, the cells were sequentially incubated with anti-desmin antibody (1 : 50) and Alexa Fluor 488 goat anti-mouse (Molecular Probes, Eugene, OR, USA). Nuclei were then visualized using Hoechst 33342 (Molecular Probes, Eugene, OR, USA). The cells were observed under an EVOS FL Digital Inverted Fluorescence Microscope (Thermo Fisher Scientific, USA). In order to determine the myogenic purity, at least 300 cells were counted in three independent cultures to obtain the percentage of desmin-positive cells. At the same time, the morphological changes of myoblasts were observed.

Tan C.M., Najib N.A., Suhaimi N.F., Halid N.A., Cho V.V., Abdullah S.I., Ismail M.Z., Khor S.C., Jaafar F, & Makpol S. (2019). Modulation of Ki67 and myogenic regulatory factor expression by tocotrienol-rich fraction ameliorates myogenic program of senescent human myoblasts. Archives of Medical Science : AMS, 17(3), 752-763.

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Temporal Dynamics of Endothelial MMP14 and Endoglin

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HUVECs were collected at different times (0, 2, 4, 6 and 8 h) after injury for the analysis of MMP14 and endoglin expression levels at the endothelial cell surface. Cells were incubated with a mouse monoclonal antibody against human endoglin (CD105; P4A4) or a rabbit monoclonal antibody against human MMP14 (ab51074, Abcam). A negative control was included using an irrelevant isotype control antibody X63 (IgG1). After washing the cells, they were incubated with Alexa Fluor 488 goat anti-mouse (A-11001 Molecular Probes, Invitrogen) for endoglin detection or Alexa Fluor 488 goat anti-rabbit IgG (11008 Molecular Probes, Invitrogen) for MMP14 detection. When working with nucleofected HUVECs, the expression levels of endoglin and MMP14 were measured by incubation with directly conjugated primary antibodies: Alexa Fluor 488 antihuman MMP14 (#128527; R&D Systems) and APC anti-endoglin/CD105 (#166707; R&D Systems). The fluorescence intensity was estimated with an EPICS XL and FC 500 flow cytometers (Beckman Coulter). A minimum of 10,000 cells were counted for each experimental point.

Gallardo-Vara E., Blanco F.J., Roqué M., Friedman S.L., Suzuki T., Botella L.M, & Bernabeu C. (2016). Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury. Angiogenesis, 19, 155-171.

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Cellular Localization of ZIP6 and ZIP7

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The cellular localization of ZIP6 and ZIP7 was determined in primary dispersed mouse islet cells and MIN6 β cells using confocal microscopy. Staining was performed as described previously (6 (link)) with primary anti-HA (1:1000, Covance Inc.), anti-FLAG (1:500, Sigma), anti-KDEL (1:200, Pierce, ThermoFisher), anti-Syntaxin-1a (1:200, Sigma), anti-ZIP7 (1:200, Proteintech, Chicago, IL), anti-ZIP6-Y3 (1:20, an antibody generated in-house by Kathryn M. Taylor, Cardiff University, UK (19 (link))), or anti-insulin (1:100, Dako) primary antibody, followed by Alexa Fluor 488 goat anti-mouse (1:500, Molecular Probes, Life Technologies), Alexa Fluor 555 donkey anti-rabbit (1:500, Molecular Probes, Life Technologies), or donkey anti-guinea pig (1:500, Jackson ImmunoResearch Laboratories) secondary antibodies. Images were acquired on Zeiss confocal microscope at ×40 magnification with an oil lens and analyzed by LSM510 (Zeiss). Colocalization of ZIP6 and ZIP7 with membrane and ER staining was analyzed and determined with Volocity software.

Liu Y., Batchuluun B., Ho L., Zhu D., Prentice K.J., Bhattacharjee A., Zhang M., Pourasgari F., Hardy A.B., Taylor K.M., Gaisano H., Dai F.F, & Wheeler M.B. (2015). Characterization of Zinc Influx Transporters (ZIPs) in Pancreatic β Cells: ROLES IN REGULATING CYTOSOLIC ZINC HOMEOSTASIS AND INSULIN SECRETION. The Journal of Biological Chemistry, 290(30), 18757-18769.

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Fluorescent Labeling Reagents for Microscopy

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Alexa Fluor 594 goat anti-mouse IgG, Alexa Fluor 488 goat anti-mouse, Alexa Fluor 594 phalloidin, and Alexa Fluor 488 phalloidin were obtained from Molecular Probes (Eugene, Oregon). 5,5′-Dithio-bis-(2-Nitrobenzoic Acid) (DTNB) and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma-Aldrich (St. Louis, MO). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Western Lightning Chemiluminescence Reagent Plus was purchased from PerkinElmer Life Sciences (Waltham, MA).

Lai C.H., Chang S.C., Chen Y.J., Wang Y.J., Lai Y.J., Chang H.H., Berens E.B., Johnson M.D., Wang J.K, & Lin C.Y. (2016). Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane. Biology Open, 5(10), 1380-1387.

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Immunofluorescence Analysis of CAR, VCAM-1, pNF-κB, and RXRα

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Confluent HUVEC were grown on glass coverslips, some of which were treated for 24 h with CITCO (3 µM) or vehicle, and then stimulated for 24 h with TNFα (20 ng/mL). The cells were then washed with PBS, fixed with 4% paraformaldehyde and blocked in PBS/1% BSA solution. Protein localization was detected by indirect immunofluorescence using the following primary antibodies: rabbit monoclonal anti-human CAR (1:50 dilution, cat# ab186869, Abcam, Waltham, MA, USA), mouse monoclonal anti-human VCAM-1 (1:50 dilution, cat# MCA907, BioRad, Barcelona, Spain), mouse monoclonal anti-human phosphorylated-NFκB (1:100 dilution, cat# 610869, BD Biosciences, Madrid, Spain) and goat polyclonal anti-human RXRα (1:50 dilution, cat#ab24636, Abcam, Waltham, MA, USA). Immunofluorescence signals were detected using the following secondary antibodies: Alexa Fluor 488 goat anti-mouse (1:1000 dilution, cat# A11001), Alexa Fluor 488 goat anti-rabbit (1:1000 dilution, cat# A11034) or Alexa Fluor 594 chicken anti-goat (1:1000 dilution, cat#A21468), all from Molecular Probes (Life Technology, Eugene, OR, USA). To confirm specificity of antibodies, isotype controls (cat# 172730; cat# ab18451, respectively, both from Abcam, Waltham, MA, USA) or secondary antibodies only were used as negative controls. Nuclei were stained with 4′,6-diamidino 2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA).

López-Riera M., Ortega R., Hueso L., Montesinos M.C., Gomez-Cabrera M.C., Sanz M.J., Real J.T, & Piqueras L. (2021). Activation of the Constitutive Androstane Receptor Inhibits Leukocyte Adhesiveness to Dysfunctional Endothelium. International Journal of Molecular Sciences, 22(17), 9267.

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