Alexa fluor 488 tagged secondary antibody

Manufactured by Thermo Fisher Scientific

Alexa Fluor 488-tagged secondary antibody is a fluorescently labeled detection reagent used in immunoassays and other immunochemical techniques. It binds to primary antibodies and emits green fluorescence upon excitation, allowing for the visualization and detection of target proteins or other biomolecules.

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Lab products found in correlation

Anti p65 antibody, by Santa Cruz Biotechnology (3 mentions) Triton x 100, by Santa Cruz Biotechnology (2 mentions) Triton x 100, by Merck Group (2 mentions) Lsm 780, by Zeiss (2 mentions) Tnf α, by Merck Group (1 mentions) Dapi nuclear counterstaining, by Southern Biotech (1 mentions) Il 1β, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Accuri c6 plus flow cytometer, by BD (1 mentions) Chamber slide, by Thermo Fisher Scientific (1 mentions) Vectashield mounting medium containing dapi, by Vector Laboratories (1 mentions) Eclipse ti s microscope, by Nikon (1 mentions) Cellrox deep red, by Thermo Fisher Scientific (1 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions) Anti a2b5 antibody, by R&D Systems (1 mentions) Lsrii flow cytometer, by BD (1 mentions) Zen lite, by Zeiss (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions)

7 protocols using alexa fluor 488 tagged secondary antibody

Suppressing NF-κB Activation in HUVECs

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Human umbilical vein endothelial cells (HUVECs) were seeded onto 12 mm glass coverslips in 24 well plates at 75,000 cells/well. After adhering, cells underwent pretreatment with 17R-RvD1 (10 nM, 100 nM) or benzo-RvD1 (10 nM, 100 nM) or vehicle for 30 minutes, followed by addition of TNFα (1 ng/mL, Sigma-Aldrich) for 2 hours. Coverslips were then washed in ice-cold phosphate buffered saline (PBS), then fixed in 4% formaldehyde and treated with 0.5% Triton X-100 (Sigma-Alrich). Blocking with 2% FBS in 0.3% Triton X-100 was performed before overnight incubation at 4°C with anti-p65 antibody (1:50; Santa Cruz Biotechnology, Dallas, TX). An Alexa Fluor 488 tagged secondary antibody (1:200, Life Technologies, Carlsbad, CA) was then used, followed by DAPI nuclear counterstaining. Images were taken with a fluorescence microscope and analyzed with ImageJ. NF-κB activation was quantified by the ratio of fluorescent intensity in the nuclear vs. cytoplasm.

Kim A.S., Werlin E.C., Kagaya H., Chen M., Wu B., Mottola G., Jan M, & Conte M.S. (2022). 17R/S-Benzo-RvD1, a synthetic resolvin D1 analogue, attenuates neointimal hyperplasia in a rat model of acute vascular injury. PLoS ONE, 17(2), e0264217.

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Immunofluorescence Staining of p65

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Cells were seeded on 8-well chamber slides (EMS, Hatfield, PA) and after treatment, were briefly rinsed in PBS and fixed with 2% paraformaldehyde for 20 min at room temperature, followed by permeabilization in ice-cold acetone (10 min at −20°C) and 1% Triton-X100 (20 min at room temperature). Cells were incubated in a humidified chamber overnight with anti-p65 antibody (Santa Cruz Biotechnology, Dallas, TX, Cat no. SC-372) at 4°C, followed by ALEXA-Fluor 488 tagged secondary antibody (Life Technologies, Carlsbad, CA) and were visualized under a fluorescence microscope. Quantitation of fluorescent signals in nucleus and cytoplasm were performed using GIMP 2.8 software (www.gimp.org).

Chatterjee A., Sharma A., Chen M., Toy R., Mottola G, & Conte M.S. (2014). The Pro-Resolving Lipid Mediator Maresin 1 (MaR1) Attenuates Inflammatory Signaling Pathways in Vascular Smooth Muscle and Endothelial Cells. PLoS ONE, 9(11), e113480.

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Investigating NF-κB Activation in RASMCs

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RASMCs were seeded onto 8-well chamber slides at 15,000 cells/well in 10% DMEM. After adhering, cells underwent pretreatment with RvD1 (10 nM; Cayman, Ann Arbor, MI) or vehicle for 30 minutes, followed by addition of IL-1β (50 ng/ml; Sigma, Aldrich, St. Louis, MO) for 2.5 hours. At the end of the experiment, slides were washed in ice-cold PBS then fixed in 4% formaldehyde and treated with 0.5% Triton X-100 (Sigma, Aldrich, St. Louis, MO). Blocking with 2% BSA (Sigma, Aldrich, St. Louis, MO) in 0.3% Triton X-100 was performed prior to overnight incubation at 4°C with an anti-p65 antibody (1:50; Santa Cruz Biotechnology, Dallas, TX). An Alexa Fluor^® 488 tagged secondary antibody (1:200; Life Technologies, Carlsbad, CA) was then used, followed by DAPI nuclear counter staining (Southern Biotech, Birmingham, AL). Images were taken with a fluorescence microscope and analyzed via ImageJ (NIH). NF-κB activation was quantified by the ratio of fluorescent intensity in the nucleus versus cytoplasm. At least 24 randomly selected cells were analyzed per well.

Wu B., Mottola G., Chatterjee A., Lance K.D., Chen M., Siguenza I.O., Desai T.A, & Conte M.S. (2016). Perivascular delivery of Resolvin D1 inhibits neointimal hyperplasia in a rat model of arterial injury. Journal of vascular surgery, 65(1), 207-217.e3.

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Integrin αvβ6 and CAR Expression Analysis

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Integrin αvβ6 expression on the surface of CCA cells was detected using mouse anti-integrin αvβ6 antibody (Clone 10D5; Merck Millipore, Burlington, MA, USA) at a dilution of 1:100. The cells were then washed and incubated with Alexa Fluor® 488-tagged secondary antibody (Thermo Fisher Scientific). CAR expression on transduced T cells was detected using anti-cMyc FIT-C-tagged antibody (clone ab1394; Abcam, Cambridge, UK). Phenotypic analysis of T cells was performed by using anti-CD3-FITC (Clone UCHT-1), anti-CD4-APC (Clone MEM-241), anti-CD8-APC (Clone UCHT-4), anti-CD16-APC (Clone 3G8), and anti-CD56-PE (Clone AB_2563925). All of these antibodies were purchased from BioLegend (San Diego, CA, USA). Flow cytometry was performed using a BD Accuri C6 Plus Flow Cytometer (BD Biosciences, San Jose, CA, USA), and the data was analyzed by using FlowJo software (FlowJo LLC, Ashland, OR, USA).

Phanthaphol N., Somboonpatarakun C., Suwanchiwasiri K., Chieochansin T., Sujjitjoon J., Wongkham S., Maher J., Junking M, & Yenchitsomanus P.T. (2021). Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells. Frontiers in Oncology, 11, 657868.

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Immunofluorescence Staining of Olig2 and Sox10

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Cells cultured on chamber slides (Thermo Fisher Scientific; cat. 154461) were washed with PBS and fixed with 4% formaldehyde at RT for 10 min. After fixation, cells were washed 3 times with PBS, permeabilized, and blocked in blocking buffer (10% FBS and 0.4% Triton X-100 in PBS) at RT for 30 min. Then the cells were incubated in the blocking buffer containing 1:200 diluted antibody against Olig2 (Abcam; cat. ab109186) or Sox10 (Abcam; cat. ab180862) at 4°C for overnight. The cells were washed 3 times with PBS and incubated in the blocking buffer containing 1:1000 diluted Alexa Fluor 488-tagged secondary antibody (Thermo Fisher Scientific; cat. A-11008) at RT for 1 h. The cells were washed 3 times with PBS and mounted in Vectashield mounting medium containing DAPI (Vector Laboratories; cat. H-1200). Fluorescent images were recorded using a Nikon Eclipse Ti-S microscope (Nikon).

Rapp-Giles B.J., Casalot L., English R.S., Ringbauer JA J.r., Dolla A, & Wall J.D. (2000). Cytochrome c3 Mutants of Desulfovibrio desulfuricans. Applied and Environmental Microbiology, 66(2), 671-677.

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Quantifying A2B5 and Oxidative Stress

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For evaluation of A2B5 expression levels, cells were detached from dishes using Accutase, pelleted by centrifugation (200 × g, 5 min), resuspended in 3% BSA/PBS containing 10 μg/mL anti-A2B5 antibody (R&D systems; cat. MAB1416), and incubated on ice for 1 h. Cells were then washed twice with PBS and incubated in 3% BSA/PBS containing 1:100 diluted Alexa Fluor 488-tagged secondary antibody (Thermo Fisher Scientific; cat. A-11001). Cells were washed 3 times with PBS, resuspended in PBS containing 1 μg/mL Hoechst 33342, and analyzed with LSR-II flow cytometer (BD Biosciences). For evaluation of endogenous oxidative stress levels, cells were incubated with 5 μM CellRox Deep Red (Thermo Fisher Scientific; cat. C10422) or vehicle (DMSO) for 30 min. Cells were then washed once with PBS, detached from dishes using Accutase, pelleted by centrifugation, resuspended in 3% BSA/PBS, and analyzed with Attune NxT flow cytometer (Life Technologies).

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Immunofluorescence Analysis of Autophagy in Two-Cell Embryos

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Embryos at the two-cell stage (21 h of culture) were fixed in 3.7% paraformaldehyde in PBS with 0.5% Triton X-100 and 0.1% PVP for 15 min at room temperature. Next, embryos were rinsed three times in PBS with 0.1% PVP, and incubated for 1 h at room temperature with a primary antibody (anti-MAP1LC3B raised in rabbit; Cat# L7543, Sigma–Aldrich). Afterward, embryos were rinsed in PBS with 0.1% PVP, and incubated for 1 h at room temperature with an Alexa Fluor 488-tagged secondary antibody raised against rabbit (Cat# A11008, ThermoFisher Scientific). Both antibodies were diluted 1:200 in PBS with 0.1% PVP. Finally, embryos were thoroughly washed in PBS with 0.1% PVP, and mounted on slides with coverslips using Prolong Gold (ThermoFisher Scientific). Embryos were evaluated by confocal microscopy (LSM 780, Zeiss, Oberkochen, Germany) at 1000x magnification. Autophagosomes were visualized at 495 and 519 nm, respectively, for excitation and emission. NZB mitochondria (previously stained with CMXRos for the photosensitization treatment) were visualized at 543 and 580–650 nm, respectively. Images were analyzed using the ZEN lite (Zeiss).

Machado T.S., Macabelli C.H., Collado M.D., Meirelles F.V., Guimarães F.E, & Chiaratti M.R. (2020). Evidence of Selection Against Damaged Mitochondria During Early Embryogenesis in the Mouse. Frontiers in Genetics, 11, 762.

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