Mouse alexafluor 647

Manufactured by Thermo Fisher Scientific

Mouse AlexaFluor 647 is a fluorescent dye that can be conjugated to antibodies or other molecules for detection in various biological applications. It has an excitation maximum at 650 nm and an emission maximum at 665 nm, allowing detection in the far-red/near-infrared region of the spectrum.

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Lab products found in correlation

Alexa fluor 488, by Thermo Fisher Scientific (5 mentions) Lsm 710, by Zeiss (2 mentions) Fluoro gel mounting medium, by Electron Microscopy Sciences (1 mentions) Clone 3f10, by Roche (1 mentions) Deltavision elite, by GE Healthcare (1 mentions) Alexa fluor 488 anti rat, by Thermo Fisher Scientific (1 mentions) Ix71 microscope, by Olympus (1 mentions) Af2085, by R&D Systems (1 mentions) Af1924, by R&D Systems (1 mentions) Alexa fluor 594, by Thermo Fisher Scientific (1 mentions) Af1979, by R&D Systems (1 mentions) Anti oct4, by STEMCELL (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Dmi8 inverted microscope, by Leica (1 mentions) Goat anti amh, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 568, by Thermo Fisher Scientific (1 mentions) Mouse anti sox9, by Abnova (1 mentions) Goat anti foxl2, by Novus Biologicals (1 mentions) Alexa fluor 647, by Thermo Fisher Scientific (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

AlexaFluor 647 goat anti-mouse secondary antibodies Anti-mouse-IgG2a-AlexaFluor 647 AlexaFluor 647-conjugated goat anti-mouse IgG secondary antibody AlexaFluor-conjugated secondary antibodies (488, 555,633, 647-conjugated anti-mouse, rabbit, goat, chicken and rat antibodies AlexaFluor 647‐conjugated anti‐mouse AlexaFluor 647 goat anti-mouse IgG (H+L) Anti-mouse IgG AlexaFluor 647 α-mouse IgG Alexafluor-647 Goat anti-mouse IgG conjugated to AlexaFluor 647 AlexaFluor 647-conjugated goat anti-mouse IgG1 secondary antibody

7 protocols using mouse alexafluor 647

Immunofluorescence Assay for Parasite Visualization

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For immunofluorescence assays, parasites were fixed in 3% (v/v) paraformaldehyde in PBS, permeabilised with 0.25% (v/v) Triton X-100 in PBS, and blocked with 2% (w/v) bovine serum albumin. Parasites were incubated in primary and fluorophore-conjugated secondary antibodies for one hour each, before being mounted in FluoroGel mounting medium (Electron Microscopy Sciences). Fluorescence images were acquired on a DeltaVision Elite system (GE Healthcare) using an inverted Olympus IX71 microscope with a × 100 UPlanSApo oil immersion lens (NA 1.40). Images were recorded using a Photometrics CoolSNAP HQ2 (link) camera, deconvolved using SoftWoRx Suite 2.0 software, and adjusted for contrast and brightness. All images were processed further with Adobe Illustrator CS6 software. Primary antibodies used in this study were monoclonal rat anti-HA (1:100 dilution; clone 3F10, Roche) and monoclonal mouse anti-TgSAG1 (1:1,000; clone TP3, Abcam, catalogue number ab8313). Secondary antibodies used in this study were anti-mouse AlexaFluor 546 (1:500; Life Technologies, catalogue number A-11030), anti-mouse AlexaFluor 647 (1:500; Life Technologies, catalogue number A-21236), and anti-rat AlexaFluor 488 (1:200; Life Technologies, catalogue number A-11006).

Rajendran E., Hapuarachchi S.V., Miller C.M., Fairweather S.J., Cai Y., Smith N.C., Cockburn I.A., Bröer S., Kirk K, & van Dooren G.G. (2017). Cationic amino acid transporters play key roles in the survival and transmission of apicomplexan parasites. Nature Communications, 8, 14455.

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Immunofluorescence Characterization of Pluripotent Cells

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Fixed cells with 4% PFA were permeabilized with PBS containing 0.3% Triton X‐100 and then blocked with 3% bovine serum albumin (BSA) (Sigma Aldrich). Human anti‐Nanog (1:1000; rabbit polyclonal, PA1‐097; Thermo Fisher Scientific), anti‐Oct4 (1:400 mouse monoclonal, 60 093; Stem Cell Technologies), anti‐brachyury (1:20 goat polyclonal, AF2085; R&D systems), anti‐Sox17 (1:20 goat polyclonal, AF1924; R&D systems) and anti‐Otx2 (1:20 goat polyclonal, AF1979; R&D systems) primary antibodies diluted in 3% BSA were incubated for 3 hours. The following secondary antibodies: goat anti‐mouse Alexa‐Fluor‐647 (A‐21235; Life Technologies), donkey anti‐goat Alexa Fluor‐594 (A‐11058; Life Technologies) and goat anti‐rabbit Alexa‐Fluor‐488 (A‐11008; Life Technologies) were used for detection. 4',6‐diamidino‐2‐phenylindole (DAPI) was used to counterstain the nuclei. The images were acquired with Leica DMi8 inverted microscope, filter cubes and software from Leica microsystems.

De Angelis M.T., Santamaria G., Parrotta E.I., Scalise S., Lo Conte M., Gasparini S., Ferlazzo E., Aguglia U., Ciampi C., Sgura A, & Cuda G. (2019). Establishment and characterization of induced pluripotent stem cells (iPSCs) from central nervous system lupus erythematosus. Journal of Cellular and Molecular Medicine, 23(11), 7382-7394.

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3D Super-Resolution Imaging of Nav1.5 and N-Cadherin

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Cells plated in 18-mm circular coverslips were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton in PBS. Blocking was done in PBS containing 2% glycine, 2% bovine serum albumin, 0.2% gelatin, and 50 mM NH₄Cl for 30 min. Primary antibodies rabbit Na_V1.5 (Sigma) (1:50) and monoclonal mouse anti-N-cadherin (BD Bioscience) (1:100) were diluted in blocking solution and incubated overnight at 4 °C. Primary antibodies were then washed with PBS and secondary antibodies were incubated for 1 h at room temperature. Secondary antibodies used were: Mouse Alexa Fluor 647 (Life Technologies) (1:5,000) and Rabbit Alexa Fluor 568 (Life Technologies) (1:20,000). SMLM imaging for 3D reconstructions was performed as described at the beginning of this Methods section (tissue sample preparation and imaging in SMLM for CLEM)30 (link). Movies containing 2,000 frames were acquired and used to reconstruct 3D super-resolved images using QuickPALM59 (link).

Leo-Macias A., Agullo-Pascual E., Sanchez-Alonso J.L., Keegan S., Lin X., Arcos T., F.e.n.g.-.X.i.a.-.L.i.a.n.g., Korchev Y.E., Gorelik J., Fenyö D., Rothenberg E, & Delmar M. (2016). Nanoscale visualization of functional adhesion/excitability nodes at the intercalated disc. Nature Communications, 7, 10342.

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Histological and Immunofluorescence Staining Protocol

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H&E histological staining was performed on 7μm-thick sagittal sections (for 12.5 dpc, 14.5 dpc and 6 weeks) according to standard protocols. For the experiments on the TESCO deletion, immunofluorescence staining was also performed on 7μm-thick sagittal sections (for TESCO) or 12 μm-thick sections (for TES), as described elsewhere [53 (link)], using (as primary and secondary antibodies, respectively): goat anti-AMH (1:500, Santa Cruz Biotechnology) and donkey anti-goat Alexa Fluor 488 (1:200, Invitrogen); mouse anti-SOX9 (1:100, Abnova) and donkey anti-mouse Alexa Fluor 647 (1:200, Invitrogen); rabbit anti-FOXL2 (1:650, generated as described by [53 (link)] and donkey anti-rabbit Alexa Fluor 647 (1:200, Invitrogen). For the experiments on the TES deletion, immunofluorescence staining was performed using rabbit anti-SOX9 (1:300, a generous gift from Francis Poulat, Institute of Human Genetics, Montpellier, France) with donkey anti-rabbit Alexa Fluor 488 (1:500, Invitrogen) and goat anti-Foxl2 (1:300, Novus) with donkey anti-goat Alexa Fluor 568 (1:500, Invitrogen). All immunofluorescence slides were also stained with 4',6-diamidino-2-phenylindole (DAPI, Molecular Probes), to visualize nuclear DNA.

Gonen N., Quinn A., O’Neill H.C., Koopman P, & Lovell-Badge R. (2017). Normal Levels of Sox9 Expression in the Developing Mouse Testis Depend on the TES/TESCO Enhancer, but This Does Not Act Alone. PLoS Genetics, 13(1), e1006520.

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Immunostaining Protocol for Vascular Smooth Muscle Cells

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VICs were fixed in 4% paraformaldehyde for 1 hr at room temperature, rinsed in PBS twice, and then permeabilized using 0.1% TritonX-100 in PBS for 2 h. Next, samples were blocked in PBS with 5% bovine serum albumin (BSA) for 2 h at room temperature to minimize non-specific protein binding. Anti-αSMA (1:200, mouse, Abcam) primary antibodies in 5% BSA were applied to samples and incubated at 4 °C overnight with gentle shaking. Primary antibodies were removed by rinsing in PBST (0.5 wt% Tween-20 in PBS) for at least 3 h with five times buffer change. Samples were then incubated at 4 °C with secondary antibodies (1:1000, goat anti-rabbit AlexaFluor 488; goat anti-mouse AlexaFluor 647, invitrogen), phalloidin (1:300, Sigma Aldrich) and DAPI (1 mg/mL; Sigma) in PBS with 1% BSA overnight. The secondary antibody solution was removed, and the samples were rinsed for 3 h with five times buffer change with PBST. All immunostained samples were stored in PBS at 4°C until imaging (LSM 710, Zeiss).

Ma H., Macdougall L.J., Rodriguez A.G., Schroeder M.E., Batan D., Weiss R.M, & Anseth K.S. (2020). Calcium Signaling Regulates Valvular Interstitial Cell Alignment and Myofibroblast Activation in Fast-relaxing Boronate Hydrogels. Macromolecular bioscience, 20(12), e2000268.

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Immunofluorescence Staining of Viral Proteins

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Cells were fixed using 4% paraformaldehyde followed by permeabilization using 0.1% Triton-x-100 (Sigma) in PBS. Cells were rinsed 3X with PBS and 0.1% Tween-20 (wash buffer) and blocked for at least 1 hour at room temperature with 1% Normal Goat Serum diluted in wash buffer (blocking buffer). Primary antibody (mouse anti-dsRNA (J2, English Scientific), 1:4000; mouse anti-flavivirus E protein (4G2), 1:2000; mouse anti-yellow fever envelope protein (clone 2D12.A), 1:2000) was diluted in blocking buffer and applied for either 1 hour at room temperature or overnight incubation at 4 °C. Following incubation, the cells were rinsed 3X with wash buffer for at least 15 minutes per wash at room temperature. Secondary antibody was diluted in blocking buffer and incubated with the cells for at least 1 hour at room temperature. In addition, Hoechst stain (Sigma) was added during incubation with the secondary antibody. Fluorescent secondary antibodies (Goat anti-mouse AlexaFluor 488, goat anti-mouse AlexaFluor 647 (Invitrogen), were used at 1:2000 dilution. Following incubation, the cells were rinsed three times with wash buffer and stored in PBS.

Barrows N.J., Anglero-Rodriguez Y., Kim B., Jamison S.F., Le Sommer C., McGee C.E., Pearson J.L., Dimopoulos G., Ascano M., Bradrick S.S, & Garcia-Blanco M.A. (2019). Dual roles for the ER membrane protein complex in flavivirus infection: viral entry and protein biogenesis. Scientific Reports, 9, 9711.

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Flow Cytometry Analysis of Cardiomyocytes

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Flow cytometry was performed as previously described (Zhou et al., 2017 (link)). Briefly, cells were trypsinized, harvested, and suspended into single cells. Then cells were fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences). Antibodies used were mouse monoclonal anti-Tnnt2 antibody (1:200, Thermo Scientific, MA5–12960), rabbit anti-GFP antibody (1:200, Thermo Scientific, A-11122), donkey anti-mouse Alexa fluor 647 (1:200, Invitrogen, A-31571) and goat anti-rabbit Alexa fluor 488 (1:200, Invitrogen, A-11008). Cells were analyzed using FACSCalibur (BD Biosciences) and FlowJo software (FLOWJO, LCC).

Hashimoto H., Wang Z., Garry G.A., Malladi V.S., Botten G.A., Ye W., Zhou H., Osterwalder M., Dickel D.E., Visel A., Liu N., Bassel-Duby R, & Olson E.N. (2019). Cardiac Reprogramming Factors Synergistically Activate Genome-wide Cardiogenic Stage-Specific Enhancers. Cell stem cell, 25(1), 69-86.e5.

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