Alexa fluor 555 conjugate

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 555 conjugate is a fluorescent dye used in various biological and research applications. It is a synthetic dye that can be covalently attached to proteins, nucleic acids, and other biomolecules, allowing them to be detected and visualized using techniques such as flow cytometry, fluorescence microscopy, and immunoassays. The core function of the Alexa Fluor 555 conjugate is to provide a bright, photostable, and pH-insensitive fluorescent signal for labeling and detection purposes.

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Lab products found in correlation

Alexa fluor 488 conjugate, by Thermo Fisher Scientific (6 mentions) Lsm 710 confocal microscope, by Zeiss (3 mentions) Sp8 upright confocal microscope, by Leica (2 mentions) C fl b 2e c, by Nikon (2 mentions) Eclipse 80i microscope, by Nikon (2 mentions) Goat anti scip, by Santa Cruz Biotechnology (2 mentions) W32464, by Thermo Fisher Scientific (2 mentions) Lsm800 confocal microscope, by Zeiss (2 mentions) Goat serum, by Agilent Technologies (1 mentions) Alexa fluor 488 donkey anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions) Prolong gold anti fade medium with dapi, by Thermo Fisher Scientific (1 mentions) Low light time lapse microscope, by Leica (1 mentions) Alexa fluor 647 goat anti rat igg h l, by Thermo Fisher Scientific (1 mentions) Primary antibody against α actinin, by Merck Group (1 mentions) Facsdiva software, by BD (1 mentions) Dapi containing mounting medium, by Vector Laboratories (1 mentions) Alexa fluor 647 conjugate, by Cell Signaling Technology (1 mentions) Pan cytokeratin, by Thermo Fisher Scientific (1 mentions) 8 well chamber slide, by Merck Group (1 mentions) Laser scanning microscope 510, by Zeiss (1 mentions)

Spelling variants of this product

Alexa Fluor 555 (1070 citations) Alexa 555 (299 citations) Alexa Fluor conjugates (56 citations) Alexa Fluors (21 citations) Alexa Fluor 488 or 555 conjugates (2 citations) Alexa Fluor 488 and 555 conjugates (2 citations)

37 protocols using alexa fluor 555 conjugate

Embryonic Cell Lineage Tracing

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Embryos still in decidua were fixed in 4% Paraformaldehyde (PFA) for two hours, before they were soaked in 30% sucrose and mounted in OCT compound. 7 µm sections were prepared using a cryostat. The sections were streptavidin/biotin blocked followed by serum blocking (PBS with 10% FCS, 0.05% Tween20 and of 10% goat serum (DAKO) for 1 hour before the sections were incubated with primary antibodies at 4 °C overnight in blocking buffer. Primary antibodies used were rabbit anti –GFP (598, polyclonal, MBL) (1/200); rat anti-mouse c-kit (553352, clone 2B8, BD Biosciences) (1/100); biotinylated rat anti-mouse Tie2 (13–5987, clone TEK4 eBioscience) (1/100). After staining, the sections were washed three times in PBST for 15 minutes each and then incubated with fluorochrome-conjugated secondary antibody at room temperature for 1 hour. These were Alexa Fluor® 488 Donkey Anti-Rabbit IgG (H + L) (A21206, Life Technologies); Alexa Fluor® 647 Goat Anti-Rat IgG (H + L) (A21247, Life Technologies); Streptavidin, Alexa Fluor® 555 Conjugate (S32355, Life Technologies). All secondary antibodies were used at 1/400 dilution. The sections were then further washed three times in PBS and mounted using Prolong Gold anti-fade medium with DAPI (Life Technologies). Images were taken using a low-light time lapse microscope (Leica) using the Metamorph imaging software and processed using ImageJ.

Stefanska M., Batta K., Patel R., Florkowska M., Kouskoff V, & Lacaud G. (2017). Primitive erythrocytes are generated from hemogenic endothelial cells. Scientific Reports, 7, 6401.

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Cardiomyocyte Identification and Quantification

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Cells isolated from day 13 EHMs were incubated with 40 μg/mL wheat germ agglutinin (WGA; Alexa Fluor 555 Conjugate, Life Technologies) for 15 minutes at 37°C, and subsequently fixed in 4% formaldehyde. Cells were then incubated with a primary antibody against α-actinin (Sigma) followed by a secondary antibody (Alexa Fluor 488 anti-mouse IgG). Samples were labeled with DAPI (1 μg/ml) and flow cytometry was performed on an LSRII (BD Biosciences). Flow cytometry results were analyzed using FacsDiva software (BD Biosciences) or Cyflogic software. Negative controls were stained only with secondary antibody and DAPI. The gating strategy used DAPI to exclude cell fragments and multicellular clumps as well as α-actinin to distinguish cardiomyocytes from non-myocytes (Figure S2). Cell number and viability were measured with the electrical current exclusion method using the CASY TT system (Roche).

Godier-Furnémont A.F., Tiburcy M., Wagner E., Dewenter M., Lämmle S., El-Armouche A., Lehnart S.E., Vunjak-Novakovic G, & Zimmermann W.H. (2015). Physiologic force-frequency in engineered heart muscle by electromechanical stimulation. Biomaterials, 60, 82-91.

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Fluorescent Labeling and Imaging of Cells

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After the CuAAC reaction, the cells were fixed with 2% formaldehyde in PBS for 30 min at room temperature. After fixation, all samples were incubated in 20 μg mL^–1 avidin–FITC in PBS at room temperature for 30 minutes. Samples were washed with PBS for six times and their wide-field fluorescence images were captured with a Nikon Eclipse 80i microscope in bright field and fluorescence modes using a 10× objective, a FITC emission filter (Nikon C-FL B-2E/C, 465–495 nm) and 400 ms of exposure time. For confocal imaging, the fixed cells were first incubated in 3 μg mL^–1 wheat germ agglutinin, Alexa Fluor 555 conjugate (Life Technologies) in PBS for 10 minutes for staining the plasma membrane. Samples were washed with PBS for 3 times and incubated in 20 μg mL^–1 avidin–FITC in PBS at room temperature for 30 minutes. Samples were washed with PBS six times and mounted on glass slide with DAPI containing mounting medium (VECTOR Laboratories). The samples were imaged with a Leica SP8 upright confocal microscope using a 63× objective and the following excitation wavelengths: 405 nm (DAPI), 496 nm (FITC), 561 nm (Alexa 555).

Li S., Wang L., Yu F., Zhu Z., Shobaki D., Chen H., Wang M., Wang J., Qin G., Erasquin U.J., Ren L., Wang Y, & Cai C. (2016). Copper-catalyzed click reaction on/in live cells †Electronic supplementary information (ESI) available: Detailed experimental procedures, characterizations and supporting data. See DOI: 10.1039/c6sc02297a Click here for additional data file.. Chemical Science, 8(3), 2107-2114.

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Multicolor Immunofluorescence Staining Protocol

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Alpha-Smooth Muscle Actin (Mouse monoclonal antibody, IgG2a, Clone: 1A4; Pierce/Invitrogen, cat#MA5-11547) was diluted to 1:200 with Ki-67 (D3B5), (Rabbit monoclonal antibody, IgG, Alexa Fluor 647 Conjugate; Cell Signaling Technology, cat#12075S) diluted to 1:400, along with Pan Cytokeratin (AE1/AE3) (Mouse monoclonal antibody, IgG1, Alexa Fluor 488 Conjugate; ThermoFisher, cat#53-9003-82), which was diluted to 1:200 in 10% Normal Goat Serum in 1% Bovine Serum Albumin in Phosphate Buffered Saline. Primary antibodies were diluted and incubated overnight at 4 °C. After incubation, secondary antibody (Goat anti-mouse monoclonal antibody, IgG2A, Alexa Fluor 555 Conjugate; Life Technologies, cat# A21137), at 1:200 dilution was applied to the slides and incubated at room temperature for one hour. After incubation slides were washed and mounted with Slowfade Gold Antifade Mountant with DAPI (Fisher Scientific, cat#S36936) in preparation for image acquisition.

Burlingame E.A., McDonnell M., Schau G.F., Thibault G., Lanciault C., Morgan T., Johnson B.E., Corless C., Gray J.W, & Chang Y.H. (2020). SHIFT: speedy histological-to-immunofluorescent translation of a tumor signature enabled by deep learning. Scientific Reports, 10, 17507.

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Apoptosis Evaluation Using TUNEL Assay

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For cell staining, cells were plated on 8-well chamber slides (Millipore, Billerica, MA) and subjected to oxidative stress treatments as described above. (1) The terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling (TUNEL) staining for apoptotic nuclei was performed using DEAD End TUNEL kit (Promega, Madison, WI) according to the manufacturer’s instruction with modification. Briefly, cells were fixed in 4% paraformaldehyde for 25 min and then treated with permeabilization solution (0.2% Triton X-100 solution in PBS) for 5 min at room temperature. Labeling reactions were performed with 100 μl of reaction buffer for 60 min at 37°C in a humidified chamber, followed by steptavidin Alexa Fluor 555 conjugate (1:400, Life Technologies, Carlsbad, CA) staining. Slides were mounted using VECTASHIELD HardSet Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). The staining was analyzed by Zeiss 510 Laser Scanning Microscope (Carl Zeiss, Thornwood, NY). Apoptosis was evaluated as the average number of positively stained cells per DAPI labeled cells.

Zhang L., Zhou M., Qin G., Weintraub N.L, & Tang Y. (2014). MiR-92a regulates viability and angiogenesis of endothelial cells under oxidative stress. Biochemical and biophysical research communications, 446(4), 952-958.

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Apoptosis Detection by PARP Cleavage

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The cells treated above were collected and blocked for 1 h in 5 % bovine serum albumin before staining with a cleaved PARP (Asp214) (D64E10) antibody for 2 h at 37 °C. The cells were then stained with an anti-rabbit IgG (H + L) F(ab′)2 fragment (Alexa Fluor® 555 Conjugate, Life Technologies, LA) antibody for 1 h followed by washing with PBS. After washing, cells were analysed by flow cytometry using the FACScan (BD Biosciences) instrument.

Liu L., Huang P., Wang Z., Chen N., Tang C., Lin Z, & Peng P. (2016). Inhibition of eEF-2 kinase sensitizes human nasopharyngeal carcinoma cells to lapatinib-induced apoptosis through the Src and Erk pathways. BMC Cancer, 16, 813.

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Fluorescent Tracing of Mouse Brain Regions

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Eight-week-old male wild-type and GAD67^EGFP mice were injected with 100 nl Cholera Toxin subunit B (Alexa Fluor 488 Conjugate or Alexa Fluor 555 Conjugate, Life Technology GmbH) or injected with 70 nl Fluorogold (0.5%, Fluorochrome). Animals were anesthetized with isoflurane, mounted in a stereotactic apparatus, and kept under isoflurane anesthesia during surgery.
For MEC injections, coordinates were 3.1 mm lateral from the midline, 0.1 mm anterior to the transverse sinus, and 1.8 mm below cortical surface; for dorsal hippocampal, 2.4 mm posterior to bregma, 2.0 mm lateral to the midline, and 1.5 below cortical surface; and for MS, 1 mm anterior to bregma and 4 mm below cortical surface.
Animals were perfused 5 to 12 days after injection and the brains processed with immunohistochemical methods. For details, see the Supplemental Experimental Procedures.

Fuchs E.C., Neitz A., Pinna R., Melzer S., Caputi A, & Monyer H. (2016). Local and Distant Input Controlling Excitation in Layer II of the Medial Entorhinal Cortex. Neuron, 89(1), 194-208.

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Cellular Imaging of Fluorescent Probes

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After the CuAAC reaction, the cells were fixed with 2% formaldehyde in PBS for 30 min at room temperature. After fixation, all samples were incubated in 20 µg/mL avidin-FITC in PBS at room temperature for 30 minutes. Samples were washed with PBS for six times and their wide-field fluorescence images were captured with a Nikon Eclipse 80i microscope in bright field and fluorescence mode using a 10× objective, a FITC emission filter (Nikon C-FL B-2E/C, 465–495 nm) and 400 ms of exposure time. For confocal imaging, the fixed cells were first incubated in 3 µg/ml wheat germ agglutinin, Alexa Fluor 555 conjugate (Life Technologies) in PBS for 10 minutes for staining the plasma membrane. Samples were washed with PBS for 3 times and incubated in 20 µg/ml avidin-FITC in PBS at room temperature for 30 minutes. Samples were washed with PBS for six times and mounted on glass slice with DAPI containing mounting medium (VECTOR Laboratories). The samples were imaged with a Leica SP8 upright confocal microscope using a 63× objective and the following excitation wavelengths: 405 nm (DAPI), 496 nm (FITC), 561 nm (Alexa 555).

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Lectin-Based Visualization of hESC and iPSC

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hESCs and iPSCs cultured on matrigel-coated coverslips for 4–5 days were incubated with wheat germ agglutinin (Alexa Fluor 555 Conjugate) (W32464; Invitrogen) and ECL (Fluorescein labeled Erythrina Cristagalli Lectin (ECL, ECA)) (FL-1141; Vector) at 1:200, for 10 minutes at room temperature. The cells were then washed three times with PBS and fixed for 20 minutes with 4% paraformaldehyde. They were washed three times with PBS and counterstained with DAPI. Imaging was performed on a Zeiss LSM510 laser scanning microscope.

Rapti K., Stillitano F., Karakikes I., Nonnenmacher M., Weber T., Hulot J.S, & Hajjar R.J. (2015). Effectiveness of gene delivery systems for pluripotent and differentiated cells. Molecular Therapy. Methods & Clinical Development, 2, 14067-.

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Quantifying Phrenic Motor Neuron Counts

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Immunohistochemistry was performed as previously described (Philippidou et al., 2012 (link); Vagnozzi et al., 2020 ), on tissue fixed for 2 hours in 4% paraformaldehyde (PFA) and cryosectioned at 16μm. Wholemounts of diaphragm muscles were stained as described (Philippidou et al., 2012 (link)). The following antibodies were used: goat anti-Scip (1:5000; Santa Cruz Biotechnology, RRID:AB_2268536), mouse anti-Islet1/2 (1:1000, DSHB, RRID:AB_2314683) (Tsuchida et al., 1994 (link)), rabbit anti-neurofilament (1:1000; Synaptic Systems, RRID:AB_887743), rabbit anti-synaptophysin (1:250, Thermo Fisher, RRID:AB_10983675), and α-bungarotoxin Alexa Fluor 555 conjugate (1:1000; Invitrogen, RRID:AB_2617152). Images were obtained with a Zeiss LSM 800 confocal microscope and analyzed with Zen Blue, ImageJ (Fiji), and Imaris (Bitplane). Phrenic MN number was quantified using the Imaris “spots” function to detect cell bodies that coexpressed high levels of Scip and Isl1/2 in a region of interest limited to the left and right sides of the ventral spinal cord.

Vagnozzi A.N., Moore M.T., de Boer R.L., Agarwal A., Zampieri N., Landmesser L.T, & Philippidou P. (2023). Catenin signaling controls phrenic motor neuron development and function during a narrow temporal window. bioRxiv.

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