Alex fluor 568

Manufactured by Thermo Fisher Scientific

Sourced in United States

AlexaFluor 568 is a fluorescent dye used in various biological and chemical applications. It has an excitation maximum at 578 nm and an emission maximum at 603 nm, making it suitable for fluorescence microscopy and flow cytometry techniques. The dye can be conjugated to a wide range of biomolecules, including antibodies, proteins, and nucleic acids, to enable detection and visualization of target analytes.

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Lab products found in correlation

700 confocal microscope, by Zeiss (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) Streptavidin horseradish peroxidase complex, by Agilent Technologies (1 mentions) M3562, by Agilent Technologies (1 mentions) Cortactin, by Thermo Fisher Scientific (1 mentions) Mrtf a, by Santa Cruz Biotechnology (1 mentions) E cadherin, by Thermo Fisher Scientific (1 mentions) Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions) Leica cryostat, by Leica Microsystems (1 mentions) In situ death detection kit, by Roche (1 mentions) Anti desmin, by Cell Signaling Technology (1 mentions) Magnafire sp digital camera, by Olympus (1 mentions) Tunel staining kit, by Roche (1 mentions) Neurolucida explorer, by MBF Biosciences (1 mentions) Slowfade diamond antifade mountant, by Thermo Fisher Scientific (1 mentions) Neurolucida 360, by MBF Biosciences (1 mentions) Lsm 880 airyscan confocal microscope, by Zeiss (1 mentions)

5 protocols using alex fluor 568

Immunohistochemical Analysis of AR-V7 in Breast Tissue

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Immunohistochemical staining for AR-V7 was done on serial 4 μm breast tissue sections as described previously [62 (link)] using the AR-V7 EPR15656 antibody (1:200), biotinylated anti-rabbit antibody (1:400, DAKO Corp., Carpinteria, CA), streptavidin-horseradish peroxidase complex (1:500, DAKO), and diaminobenzidine tetrahydrochloride. To analyze 22Rv1 cells (Supplementary Figure S4), a standard cytospin protocol was utilized [63 (link)] followed by immunohistochemistry as above.
Preparation of formalin-fixed, paraffin-embedded tissue sections for immunofluorescence was done as described previously [64 (link)]. For all antigens, retrieval was performed in 600 mL of 10 mM Tris base and 1 mM Na-EDTA (pH 9.0) by heating in a 1100W microwave at full power for 5 min and subsequently heating at 50% power for an additional 5 min. Primary antibodies used for immunofluorescence were AR-441 (M3562, 1:50, DAKO) and AR-V7 (EPR15656, 1:400). Primary antibodies were detected using secondary antibodies conjugated to either Alexa-Fluor 488 (A11029; Life Technologies) or Alex-Fluor 568 (A11036; Life Technologies). Images were acquired sequentially on a Zeiss 700 confocal microscope with a pinhole aperture of 2 airy units.

Hickey T.E., Irvine C.M., Dvinge H., Tarulli G.A., Hanson A.R., Ryan N.K., Pickering M.A., Birrell S.N., Hu D.G., Mackenzie P.I., Russell R., Caldas C., Raj G.V., Dehm S.M., Plymate S.R., Bradley R.K., Tilley W.D, & Selth L.A. (2015). Expression of androgen receptor splice variants in clinical breast cancers. Oncotarget, 6(42), 44728-44744.

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Analyzing TGF-β2-Mediated Cytoskeletal Dynamics

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Recombinant human TGF-β2 (TGF-β) was purchased from R&D Systems (Minneapolis, MN, USA). MMP9-specific inhibitor JNJ0966 was purchased from Tocris (Bristol, UK). Primary antibodies that were used include αSMA conjugated to fluorescein isothiocyanate (FITC) from Sigma Aldrich (Oakville, ON, Canada), FAK from Abcam (Waltham, MA, USA), E-cadherin, pFAK at Tyr397, LIMK1, pLIMK1 at Thr508 and cortactin from Invitrogen (Waltham, MA, USA), pMLC2 at Ser18 from Millipore Sigma (Burlington, MA, USA), and MRTF-A from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies for immunofluorence staining were obtained from Molecular probes (Invitrogen, Carlsbad, CA, USA), and phalloidin conjugated to Alex Fluor^®568 was obtained from Life Technologies (Eugene, OR, USA).

Liu Z.Z., Taiyab A, & West-Mays J.A. (2021). MMP9 Differentially Regulates Proteins Involved in Actin Polymerization and Cell Migration during TGF-β-Induced EMT in the Lens. International Journal of Molecular Sciences, 22(21), 11988.

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TUNEL Staining of DNA Strand Breaks

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Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining of DNA strand breaks was performed using the In Situ Death Detection Kit (Roche, Branchburg, NJ, USA), as previously described (55 (link)). Fresh frozen heart sections were cut using a Leica cryostat (Model CM3050S, Leica Microsystems) to produce 7-µm tissue sections. Tissue sections were fixed with 4% paraformaldehyde for 20 min and permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate for 2 min at 4°C. To distinguish myocytes from nonmyocytes in the heart, tissue sections were incubate with anti-Desmin (Cell Signaling, 1:100) followed by incubation with anti-Rabbit secondary conjugate with Alex Fluor 568 (Invitrogen). Fifty microliters of a reaction mixture containing terminal deoxynucleotidyl transferase (TdT), fluorescein-dUTP was added to each section and incubated in a humidified chamber for 60 min at 37°C. Sections were washed three times with PBS and counterstained with 4’,6’-diamidino-2-phenylindole (DAPI, 5 µg/ml) for 1 min. Slides were mounted with the Prolong Gold mounting medium (Invitrogen, Carlsbad, CA, USA), and five images per tissue section were obtained using a Zeiss confocal microscope equipped with an Olympus MagnaFire SP digital camera and ImagePro image analysis software as previously described (56 (link)).

Wang Q, & Ren J. (2016). mTOR-Independent Autophagy Inducer Trehalose Rescues against Insulin Resistance-Induced Myocardial Contractile Anomalies: Role of p38 MAPK and Foxo1. Pharmacological research, 111, 357-373.

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Detecting Myocardial Apoptosis Using TUNEL Assay

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Frozen myocardial sections were obtained from the mid‐section of the whole heart (about half way between apex and mitral valves) and were stained with a TUNEL staining kit (Roche). Sections were rinsed in PBS buffer for 10 minutes before incubation with proteinase K (20 μg/mL) in Tris‐HCl for 30 minutes at 37°C. In a humidified chamber, sections were incubated with the anti‐Desmin antibody (Cell Signaling, 1:50) at 4°C overnight. After that, sections were incubated with anti‐Rabbit secondary conjugate with Alex Fluor 568 (Invitrogen) at 37°C for 1 hour. Sections were then incubated with the TUNEL staining solution at 37°C for 1 hour in a humidified chamber. Following PBS wash, myocardial sections were stained with DAPI for 10 minutes at 37°C in a humidified chamber. Sections were mounted with 50% glycerol and were used for fluorescent detection using confocal microscopy. The percentage of apoptotic cells in total cells was calculated.33, 34

Yang L., Ma J., Tan Y., Zheng Q., Dong M., Guo W., Xiong L., Yang J, & Ren J. (2019). Cardiac‐specific overexpression of metallothionein attenuates L‐NAME‐induced myocardial contractile anomalies and apoptosis. Journal of Cellular and Molecular Medicine, 23(7), 4640-4652.

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Neuronal Morphology Characterization in Transgenic Mice

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Brain slices from slice electrophysiology were subjected to histochemical analysis using NEUN antibody to confirm neuron identity and streptavidin Alex Fluor-568 (Invitrogen) to label injected biocytin for morphology assessment. Stained sections were mounted in cell gasket with SlowFade Diamond Antifade Mountant (Invitrogen). Images for neuronal body and dendrites were taken under Zeiss LSM 880 Airyscan Confocal Microscope. We used Neurolucida 360 (https://www.mbfbioscience.com/neurolucida360) to trace the neuronal body (15 neurons from 8 WT animals, 14 neurons from 8 Het animals) and dendrites (10 neurons from 5 WT animals, 10 neurons from 6 Het animals) and count different types of dendritic spines (10 neurons from 4 WT animals, 7 neurons from 4 Het animals). Branch analysis and Sholl analysis were performed using Neurolucida Explorer (https://www.mbfbioscience.com/neurolucida-explorer). Then we exported measurements for soma surface area, soma volume, total dendrite number, total dendritic length, average dendrite length, dendrite node number, and complexity ([Sum of the terminal orders + Number of terminals] * [Total dendritic length / Number of primary dendrites]), branch number, branch length, total spine density, and density of different spine subtypes to compare neuron morphological maturation between Hets and WTs.

Chen J., Lambo M.E., Ge X., Dearborn J.T., Liu Y., McCullough K.B., Swift R.G., Tabachnick D.R., Tian L., Noguchi K., Garbow J.R., Constantino J.N., Gabel H.W., Hengen K.B., Maloney S.E, & Dougherty J.D. (2021). A MYT1L syndrome mouse model recapitulates patient phenotypes and reveals altered brain development due to disrupted neuronal maturation. Neuron, 109(23), 3775-3792.e14.

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