Alexa flour 488 or 594

Manufactured by Thermo Fisher Scientific

Alexa Fluor 488 and Alexa Fluor 594 are fluorescent dyes commonly used in biological and biochemical research. They emit green and red fluorescence, respectively, when excited by appropriate wavelengths of light. These dyes can be used to label various biomolecules, such as proteins, nucleic acids, or other cellular components, for detection and visualization in various analytical techniques like fluorescence microscopy, flow cytometry, and immunoassays.

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Alexa Flour 488- or 594-conjugated secondary antibodies Alexa 488 or 594 Alexa Flour 594 Alexa Flour 488 Alexa Flours Alexa 594 Alexa 488

2 protocols using alexa flour 488 or 594

Immunofluorescence Staining of 5mC and 5hmC

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For 5mC and 5hmC immunofluorescence staining, chromosomes fixed on slides were incubated in 4 N HCl at 37 °C for 30 min. After neutralization in 100 mM Tris–HCl (pH 7.5) for 10 min, the specimens were incubated with anti-5mC (mouse monoclonal; Calbiochem) and/or anti-5hmC (rabbit polyclonal; Active Motif) antibodies. After blocking with 1 % bovine serum albumin and 0.2 % Triton X-100 in phosphate-buffered saline, the specimens were incubated with anti-mouse and/or anti-rabbit secondary antibodies coupled with Alexa Flour 488 or 594 (Life Technologies) at room temperature for 30 min. The samples were then counterstained with DAPI in the mounting medium.

Kubo N., Toh H., Shirane K., Shirakawa T., Kobayashi H., Sato T., Sone H., Sato Y., Tomizawa S.I., Tsurusaki Y., Shibata H., Saitsu H., Suzuki Y., Matsumoto N., Suyama M., Kono T., Ohbo K, & Sasaki H. (2015). DNA methylation and gene expression dynamics during spermatogonial stem cell differentiation in the early postnatal mouse testis. BMC Genomics, 16(1), 624.

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Anti-Inflammatory Signaling Pathway Assays

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Fisetin (98% pure), β-actin antibody and MPO fluorometric activity assay kit were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies for F4/80, Ly-6G (Gr-1), EP1, EP2, EP3, EP4, COX-2, TNFα, IL-1β, IL-6, Cyclin D1, PCNA, p21, p53, PI3Kp110α, PI3K/p85, pAKT Ser⁴⁷³, NFκB p65, IKKα/β, IκBα, phospho-IκBα and lamin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Horseradish peroxidases conjugated anti-mouse, anti-goat and anti-rabbit secondary antibodies were purchased from Millipore Corporation (Billerica, MA). Anti-goat and anti-rabbit secondary antibodies labelled with either Alexa Flour 488 or 594 were purchased from Life Technologies Corporation (Grand Island, NY). PGE₂ EIA kit was purchased from Cayman Chemicals Company (Ann Arbor, MI). Reagents for protein estimation were purchased from Bio-Rad Laboratories (Hercules, CA). HyBlot CL and autoradiography films were procured from Denville Scientific Inc. (Metuchen, NJ).

Pal H.C., Athar M., Elmets C.A, & Afaq F. (2014). Fisetin inhibits UVB-induced cutaneous inflammation and activation of PI3K/AKT/NFκB signaling pathways in SKH-1 hairless mice. Photochemistry and photobiology, 91(1), 225-234.

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