Alexa fluor 488 secondary antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Canada, Italy, Japan, Sweden

Alexa Fluor 488 secondary antibody is a fluorescent dye-conjugated antibody used for detection and visualization in various immunoassay techniques. It binds to the Fc region of primary antibodies, allowing for amplification of the fluorescent signal and indirect labeling of target proteins or cells.

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422 protocols using alexa fluor 488 secondary antibody

Quantification of DNA Damage Markers

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Two-days after irradiation, cells grown on glass coverslips were fixed using 4% p-formaldehyde for 10 minutes, permeabilized with 0.25% Triton X-100 at room temperature and blocked with 3% BSA/PBS for 45 minutes. Cells were incubated overnight with an anti-γ-H2AX antibody (1:1000, Millipore, Temecula, CA, 05-636), anti-phospho Histone H3 (Ser10, 1:1000, Millipore, Temecula, CA, 06-570) or anti-α-tubulin (1:5000, ThermoFisher Scientific, Illinois, USA, PA5-22060) prepared in 0.05% Triton X-100 and 1% BSA/PBS. After washing, cells were incubated with an anti-mouse Alexa Fluor-488 secondary antibody (1:500, Molecular Probes, A-21042) or anti-rabbit Alexa Fluor-488 secondary antibody (1:500, Invitrogen A-11034). Slides were mounted with ProLong Gold Antifade Reagent with DAPI (Life Technologies, NY). Cells were photographed under a fluorescence microscope (BX53; Olympus, Japan). For γ-H2AX quantification, foci were counted using a Find Maxima plugin and normalized by nuclei numbers using ImageJ software (Rasband, National Institutes of Health, USA). Every focus bigger than 0.1 μm (13 pixels) was considered as positive.

Andaur R., Tapia J.C., Moreno J., Soto L., Armisen R, & Marcelain K. (2018). Differential miRNA expression profiling reveals miR-205-3p to be a potential radiosensitizer for low- dose ionizing radiation in DLD-1 cells. Oncotarget, 9(41), 26387-26405.

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Immunofluorescence Staining Protocol

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Cells were plated on a standard multiwell culture plate to reach confluence the following day. Cells were rinsed with filtered 1X PBS, fixed with ice-cold methanol for 10 min, washed three times with PBS, blocked with 5% filtered bovine serum albumin (BSA) in PBS, then probed overnight at 4°C with respective primary antibodies in 5% BSA-PBS solution. Horse serum (RMBIO DES-BBT) diluted to 30% in PBS was used as blocking solution for pro-SFTPC antibody. The following day, cells were washed with 1X TBST (20 mM Tris, 150 mM NaCl, 0.01% Tween 20, pH 7.5), probed in PBS with biotinylated secondary antibodies for 1 h, washed, then probed with Streptavidin-Alexa Fluor 647 conjugate (ThermoFisher S21374). For double staining, cells were blocked again, then probed with either donkey anti-rabbit Alexa Fluor 488 secondary antibody (ThermoFisher A21206) or donkey anti-mouse Alexa Fluor 488 secondary antibody (ThermoFisher A21202). Mounting solution with 4′,6-diamidino-2-phenylindole (DAPI) was used as nuclear counterstain (Vector Laboratories H-1200). Stained cells were viewed using Nikon Eclipse Ti-U inverted fluorescence microscope and imaging software, NIS-Elements Br (v4.00.12, build 802, 64-bit).

Tran E., Shi T., Li X., Chowdhury A.Y., Jiang D., Liu Y., Wang H., Yan C., Wallace W.D., Lu R., Ryan A.L., Marconett C.N., Zhou B., Borok Z, & Offringa I.A. (2022). Development of human alveolar epithelial cell models to study distal lung biology and disease. iScience, 25(2), 103780.

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Quantifying Cell Adhesion Molecules

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The expression of VCAM‐1 and ICAM‐1 was measured by flow cytometry, as previously described (Azcutia et al., 2010). Primary antibodies against VCAM‐1 (clone IE5; Chemicon, Temecula, CA) or ICAM‐1 (clone 6.5B5; Chemicon) were used at a 1/100 dilution, followed by incubation with an appropriate Alexa Fluor 488 secondary antibody (Molecular Probes, Invitrogen Corporation, Carlsbad, CA; dilution 1/250). Fluorescence was measured in a FACScan flow cytometer (Beckton‐Dickinson, Franklin Lakes, NJ), and data were analyzed using CXP analysis software (Beckton‐Dickinson).

Romero A., San Hipólito‐Luengo Á., Villalobos L.A., Vallejo S., Valencia I., Michalska P., Pajuelo‐Lozano N., Sánchez‐Pérez I., León R., Bartha J.L., Sanz M.J., Erusalimsky J.D., Sánchez‐Ferrer C.F., Romacho T, & Peiró C. (2019). The angiotensin‐(1‐7)/Mas receptor axis protects from endothelial cell senescence via klotho and Nrf2 activation. Aging Cell, 18(3), e12913.

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Visualization of UV-induced Polη, Polκ, and Rev7 foci in MEFs

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To examine UV-induced Polη and Polκ focus formation in MEFs, Rev1^+/+ and Rev1^−/− MEFs were immortalized by lentiviral expression of SV40-T antigen (GeneCopoeia). GFP-Polη or GFP-Polκ was transiently transfected into transformed MEFs. After 16 h of incubation, cells were suspended and cultured on a coverslip in a six-well plate with 50% confluence. After 48 h, cells were treated with 20 J/m² of UVC. After 6 h of incubation, cells were fixed with 4% paraformaldehyde for 30 min. Nuclear staining was performed with DAPI (Molecular Probe) in PBS buffer for 20 min. To examine Rev7 foci in MEFs, primary Rev1^+/+ and Rev1^−/− MEFs were cultured on a coverslip and incubated for 20 h. Cells were treated with 20 J/m² of UVC, and, after 6 h incubation, cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were permeablized with 0.2% Triton X-100 in PBS buffer. Cells were immunostained with Rev7 antibody (BD Bioscience) and then incubated with Alexa fluor 488 secondary antibody (Molecular Probe). Nuclear DNA was stained with DAPI (Molecular Probe) for 20 min. The fluorescent images were visualized and captured by fluorescence microscope (Nikon fluorescence microscope).

Yoon J.H., Park J., Conde J., Wakamiya M., Prakash L, & Prakash S. (2015). Rev1 promotes replication through UV lesions in conjunction with DNA polymerases η, ι, and κ but not DNA polymerase ζ. Genes & Development, 29(24), 2588-2602.

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Visualization of Vimentin Filaments in Cultured Cells

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After 24h seeding, cultures were fixed with 4% paraformaldehyde in PBS (SIGMA-ALDRICH, St. Louis, MO) at RT for 20 min. Samples were blocked and permeabilized in 10% bovine serum albumin and 0.1% Triton-X 100 for 10 and 5 minutes, respectively, and afterwards incubated for 1h with primary mouse monoclonal antibodies directed against vimentin (1:200, SIGMA-ALDRICH, St. Louis, MO). After rinsing, the following stains were used: rhodamine-phalloidin (1:500, Molecular Probes, Eugene, OR) was used to label actin filaments, Hoechst (1:1000, Molecular Probes, Eugene, OR) was used to label cell nuclei and anti-mouse Alexa Fluor 488 secondary antibody (1:500, Molecular Probes, Eugene, OR) was used for vimentin filament visualization. The cells were examined using a Leica DMIRE2 inverted microscope (Leica, Buffalo Grove, IL) and images were acquired using a Hamamatsu camera (Hamamatsu, Japan). Immunofluorescence was recorded with a 100x oil lens with a numerical aperture of 1.25. In order to quantify cell adherent area, a 40x air lens with a numerical aperture of 0.60 and phase contrast was used. For each sample, a minimum of 20 images was acquired from different locations and approximately 100 cells per substrate were analyzed. Cell area was calculated using ImageJ software by precise tracing of single cell outlines (Image J Software, NIH, Bethesda, MD) ³³.

Pogoda K., Chin L., Georges P.C., Byfield F.J., Bucki R., Kim R., Weaver M., Wells R.G., Marcinkiewicz C, & Janmey P.A. (2014). Compression stiffening of brain and its effect on mechanosensing by glioma cells. New journal of physics, 16, 075002-.

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Intracellular Localization of Hexokinase

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Intracellular localization of HK was studied on cells cultured on glass coverslips and treated with MitoTracker probe (Life Technologies Ltd, Monza MB, Italy), rabbit anti-HKII (C64G5) (primary antibodies (Euroclone) and then with a goat anti-Rabbit Alexa Fluor 488 secondary antibody (Molecular Probes Eugene, OR, USA). Results were analyzed using an Olympus (Olympus Optical) laser-scanning microscope FV500 equipped with an Olympus IX81 inverted microscope and Argon ion 488 nm, He-Ne 543 nm, and He-Ne 633 nm lasers. Digital images were acquired through a PLAPO 60 × objective, with the Fluoview 4.3b software program. Images were acquired sequentially as single transcellular optical sections. Spatial co-localization was analyzed by Image J 1.34f software (NIH).

Marini C., Bianchi G., Buschiazzo A., Ravera S., Martella R., Bottoni G., Petretto A., Emionite L., Monteverde E., Capitanio S., Inglese E., Fabbi M., Bongioanni F., Garaboldi L., Bruzzi P., Orengo A.M., Raffaghello L, & Sambuceti G. (2016). Divergent targets of glycolysis and oxidative phosphorylation result in additive effects of metformin and starvation in colon and breast cancer. Scientific Reports, 6, 19569.

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Immunolabeling of Phosphorylated Signaling Proteins

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Embryos and tissue explants were fixed in either 4% PFA or 10% Neutral Buffered Formalin (NBF) for one hour on ice. The samples were embedded and cryosectioned as described. The immunolabeling protocol was the same as the cell shape visualization protocol described above except that the incubation time with the primary antibodies ranged from overnight (pSmad2 and pERK) to four days (pSmad1/5/8) at 4°C. For pERK staining, antigen retrieval was performed by microwaving in Antigen Unmasking solution (H-3301, Vectorlabs). The following antibodies were used: primary antibodies used were pSmad2 (1:25, rabbit polyclonal, 3101, Cell Signaling Technology, RRID:AB_331673), pSmad1/5/8 (1:100, rabbit polyclonal, 9511, Cell Signaling Technology, RRID:AB_331671), pERK (1:5, rabbit polyclonal, 9101, Cell Signaling Technology, RRID:AB_2315113); and the secondary antibody was Rabbit Goat anti-rabbit Alexa-Fluor 488 secondary antibody (1:400, A-11008, Molecular Probes).

Ray P, & Chapman S.C. (2015). Cytoskeletal Reorganization Drives Mesenchymal Condensation and Regulates Downstream Molecular Signaling. PLoS ONE, 10(8), e0134702.

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Immunocytochemistry Analysis of Oxidative Stress

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For immunocytochemistry analysis, 1 × 10⁴ human corneal endothelial cells were seeded on coverslips and transfected with control or SLC4A11 siRNA as described above. The cells were then exposed to oxidative stress, washed in PBS and fixed in 4% paraformaldehyde for 15 min. For immunofluorescence analysis of pIkB, MAPK, Nitrotyrosine, or cytochrome c, staining was done as described^{53 (link)}. Cells were stained with rabbit anti-pERK, anti-pJNK, anti-pp38 or anti-pIkB antibodies (1:100, Cell Signaling Technology, Beverly, MA), mouse anti-nitrotyrosine (1:100, Novus Biologicals, Littleton, CO), or mouse anti-cytochrome c antibody (1:100, BioLegend, SanDiego, CA) for 45 min followed by Alexafluor 488 secondary antibody (1:500, Molecular probes, Eugene, OR) for 30 min. Images were captured on a fluorescent microscope (Olympus IX73) using a 20X objective. The fluorescence intensities were measured by ImageJ software.

Guha S., Chaurasia S., Ramachandran C, & Roy S. (2017). SLC4A11 depletion impairs NRF2 mediated antioxidant signaling and increases reactive oxygen species in human corneal endothelial cells during oxidative stress. Scientific Reports, 7, 4074.

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Immunostaining of Phospho-Histone H2AX

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The culture media was replaced with pre-warmed media. The cells were kept in a CO₂ incubator at 37°C for 15 min and then fixed with 4% paraformaldehyde for 15 min at room temperature. Subsequently, the samples were immunostained [6 (link)] with anti-phospho-histone H2AX (Ser139) clone JBW301 antibody (05-636, Merck Millipore, MA) and Alexa Fluor^®488 secondary antibody (A-11001, Molecular Probes, OR).

Kodaira S., Konishi T., Kobayashi A., Maeda T., Ahmad T.A., Yang G., Akselrod M.S., Furusawa Y, & Uchihori Y. (2014). Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector. Journal of Radiation Research, 56(2), 360-365.

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Antibody Responses to Bacterial Antigens

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C57BL/6 mice were immunized with 10 µg of either OVA_323–339, MPO_409–428, S. aureus pSJH101-derived 6PGD_391–410, S. aureus Variant 1 6PGD_391–410, S. aureus Variant 2 6PGD_391–410, S. aureus Variant 3 6PGD_391–410 or S. aureus Variant 4 6PGD_391–410; first on day 0 emulsified in (FCA), then boosted on days 7 and 14 emulsified in Freund’s incomplete adjuvant (FIA). Serum was collected from mice by cardiac puncture on day 28 and Protein G purified for indirect immunofluorescence on ethanol fixed neutrophils. Thioglycolate induced peritoneal neutrophils were obtained from either Mpo^+/+ or Mpo^−/− C57BL/6 mice, cytospun onto glass slides then ethanol fixed^{6 (link),33 (link)}. Pooled serum IgG was incubated with slides for 1 h, then anti-mouse IgG detected using a chicken anti-mouse Alexa Fluor 488 secondary antibody (Molecular Probes, A-21200, 1:200). DAPI was used as a nuclear stain and fluorescence detected by either fluorescence microscopy or confocal microscopy.

Ooi J.D., Jiang J.H., Eggenhuizen P.J., Chua L.L., van Timmeren M., Loh K.L., O’Sullivan K.M., Gan P.Y., Zhong Y., Tsyganov K., Shochet L.R., Ryan J., Stegeman C.A., Fugger L., Reid H.H., Rossjohn J., Heeringa P., Holdsworth S.R., Peleg A.Y, & Kitching A.R. (2019). A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity. Nature Communications, 10, 3392.

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