Dylight 488 streptavidin

Manufactured by Vector Laboratories

Sourced in United States, France

DyLight 488 Streptavidin is a fluorescent reagent used in various biomedical applications. It consists of the protein streptavidin conjugated to the DyLight 488 fluorescent dye. DyLight 488 is a green-fluorescent dye with excitation/emission maxima of 493/518 nm.

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Lab products found in correlation

Lsm 710, by Zeiss (4 mentions) Biotinylated goat anti rabbit igg, by Vector Laboratories (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) Rabbit anti flash, by Merck Group (2 mentions) Rabbit anti lsm10, by Merck Group (2 mentions) Rabbit anti gaf, by Merck Group (2 mentions) Mouse anti laci, by Merck Group (2 mentions) Mouse anti mpm 2, by Merck Group (2 mentions) Alexa fluor secondary antibody, by Thermo Fisher Scientific (2 mentions) 4 6 diamino phenylindole dapi, by Merck Group (1 mentions) Las af tcs sp8, by Leica Microsystems (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Leica tcs sp8 confocal microscope, by Leica Microsystems (1 mentions) B 1355, by Vector Laboratories (1 mentions) Pha l, by Vector Laboratories (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Anti p akt, by Cell Signaling Technology (1 mentions) Sorafenib, by MedChemExpress (1 mentions) Anti smad2, by Cell Signaling Technology (1 mentions) Anti smad3, by Cell Signaling Technology (1 mentions)

24 protocols using dylight 488 streptavidin

Immunofluorescence Analysis of Stem Cell Markers

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NT2/D1 cells were plated on coverslips and cultured in the absence (for 2 days) or presence (for 2, 4 and 7 days) of RA. Following fixation in 4% paraformaldehyde for 20 min at room temperature and permeabilization in 0.1% Triton X 100, cells were incubated in blocking solution, 10% normal goat serum and 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. Primary antibodies diluted in 1% BSA (Sigma Aldrich, USA), 0.1% Triton X 100 in PBS were applied overnight at 4°C as follows: mouse monoclonal anti OCT-3/4 (sc-5279, diluted 1: 100; Santa Cruz Biotechnology, Inc) and rabbit polyclonal anti SOX3 (sc-20089, diluted 1: 100; Santa Cruz Biotechnology, Inc). Coverslips were washed three times for 10 min in 0.1% Triton X 100 in PBS and incubated with biotinylated goat anti rabbit IgG (1: 500; Vector, USA) for 1 h at room temperature in 1% BSA, 0.1% Triton X 100 in PBS followed by DyLight 488^® streptavidin (1: 1000; Vector Laboratories, USA) and Alexa FluorH 594, (1: 500; InvitrogenTM) diluted in PBS for 1 h at room temperature. Nuclei were stained with 0.1 mg/ml 4′,6 diamino phenylindole (DAPI; Sigma Aldrich). Samples were viewed and images were taken using a Leica TCS SP8 confocal microscope and Leica Microsystems LAS AF TCS SP8 software (Leica Microsystems, Germany).

Topalovic V., Krstic A., Schwirtlich M., Dolfini D., Mantovani R., Stevanovic M, & Mojsin M. (2017). Epigenetic regulation of human SOX3 gene expression during early phases of neural differentiation of NT2/D1 cells. PLoS ONE, 12(9), e0184099.

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Co-Staining of MMP13 and PNN in Rat Brains

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Co-staining for MMP13 and PNN was done as described in (Rankin-Gee et al., 2015 (link)) with slight modifications. Rats were perfused with 4% paraformaldehyde via cardiac infusion. Brains were removed and kept in 4% paraformaldehyde overnight before being transferred to a 30% sucrose solution. Forty micrometer brain slices were collected using a microtome and were stored in cryopreservative. Slices were washed with PBS five times for 5 min each. Slices were blocked in PBS + 2% BSA for 1 h at room temperature. Sections were co-incubated in biotinylated Wisteria Floribunda Lectin (WFA) (20 μg/ml, B-1355, Vector Labs) and anti-MMP13 antibody (1:100, MAB13426, EMD Millipore Corp., CA) in PBS + 2% BSA overnight. Sections were incubated in DyLight 488 Streptavidin (1:500, SA-5488–1, Vector Labs) and 594 Alexa fluorescent-conjugated goat anti-mouse antibody for 3 h at room temperature after washes in PBS. Slices were then mounted with Vectashield mounting medium after staining with DAPI. Confocal images of stained sections were captured using Leica Microsystems, IL, USA with a 40X magnification oil lens. Images were processed in ImageJ.

Dubey D., McRae P.A., Rankin-Gee E.K., Baranov E., Wandrey L., Rogers S, & Porter B.E. (2017). Increased metalloproteinase activity in the hippocampus following status epilepticus. Epilepsy research, 132, 50-58.

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Immortalized Hepatic Stellate Cell Assay

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LX-2 cells, an immortalized hepatic stellate cell line^{47 (link)}, were cultured in Dulbecco’s modified Eagle medium (DMEM) plus 10% fetal bovine serum (FBS) which was kindly provided by Dr. Yi-Tsau Huang (National Yang-Ming University, Taipei, Taiwan). Antibodies including anti-p-Erk1/2, anti-p-Akt, anti-Erk1/2, anti-Akt, anti-p-smad2, anti-p-smad3, anti-smad2, and anti-smad3 were from Cell Signaling. Anti-neuropilin (NRP)-1 and α-smooth muscle actin (α-SMA) antibodies were from GeneTex and Millipore. The polyclonal anti-Gal-1 antibody was purified from Gal-1-immunized rabbit serum. Recombinant PDGF and TGF-β were from R&D Systems. Inhibitors of N- and O-glycosylation [swainsonine (SW) and benzyl-N-acetyl-α-galactosaminide (BαG)] were from Calbiochem. Sorafenib and SIS3 were from MedChemexpress (MCE). Biotinylated lectins including L-PHA, LEL, SNA, PNA and DyLight® 488 streptavidin were from Vector Labs. Thiodigalactoside (TDG) was from Carbosynth.

Wu M.H., Chen Y.L., Lee K.H., Chang C.C., Cheng T.M., Wu S.Y., Tu C.C, & Tsui W.L. (2017). Glycosylation-dependent galectin-1/neuropilin-1 interactions promote liver fibrosis through activation of TGF-β- and PDGF-like signals in hepatic stellate cells. Scientific Reports, 7, 11006.

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Brain Tissue Immunostaining Protocol

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Formalin-fixed, paraffin-embedded SWS brain sections were deparaffinized and immersed in antigen retrieval solution (Citrate-EDTA buffer: 10 mM citric acid, 2 mM EDTA, 0.05% Tween 20, pH 6.2 or 1mM EDTA pH 8.0) for 20 minutes. Sections were blocked for 30 minutes and stained with anti–human VE-Cadherin antibody (Sigma, HPA030562), anti-human CD31 antibody (Santa Cruz, C-20) and anti-human VEGFR2 antibody (55B11, Cell Signaling Technology, Danvers, MA) overnight. Purified class- and species-matched IgGs (Vector Lab) were the controls. Afterwards, sections were incubated with appropriate biotinylated secondary antibodies followed by DyLight 488 Streptavidin (Vector Lab). All slides were mounted using DAPI (Molecular Probe, Eugene, OR) to visualize nuclei. Images were acquired using a Leica TCS sp2 Acousto-Optical Beam Splitter confocal system equipped with DMIRE2 inverted microscope camera (Leica Microsystems, Wetzlar, Germany).

Huang L., Couto J.A., Pinto A., Alexandrescu S., Madsen J.R., Greene A.K., Sahin M, & Bischoff J. (2016). Somatic GNAQ mutation is enriched in brain endothelial cells in Sturge-Weber Syndrome. Pediatric neurology, 67, 59-63.

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Immunohistochemical Staining Reagents

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Primary antibodies raised against pERK1/2 and ERK 1 were obtained from Cell Signaling Technology (Leiden, Netherlands) and Santa Cruz Biotechnology (Heidelberg, Germany) respectively. Rabbit anti-tryptophane-5-hydroxylase antibodies (TPH) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit and mouse anti-glial fibrillary acidic protein antibodies (GFAP) were from Dako and Sigma. Anti-rabbit and anti-mouse biotinylated secondary antibodies were obtained from Vector laboratories (Nanterre, France). The streptavidin-peroxidase conjugate was purchased to Alpha Diagnostic International (Texas, USA) and Santa Cruz Biotechnology (Heidelberg, Germany). DyLight 488 Streptavidin was from Vector laboratories (Nanterre, France). Texas Red anti-rabbit secondary antibodies were obtained from Jackson Immunoresearch Laboratories (Pennsylvania, USA).

Lépinay J., Taragnat C., Dubois J.P., Chesneau D., Jockers R., Delagrange P, & Bozon V. (2021). Negative regulation of melatonin secretion by melatonin receptors in ovine pinealocytes. PLoS ONE, 16(7), e0255249.

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Immunocytochemistry for Apoptosis and β-Catenin

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Immunocytochemistry for CC3 and β‐catenin was performed to analyse cell death after 24‐hr stimulation with Wnt ± H₂O₂ or to analyse β‐catenin localization after 30 min. VSMCs were fixed with 3% paraformaldehyde/PBS and permeabilized with 0.1%–0.2% Triton X‐100/PBS. After blocking with 20% goat serum/PBS, 1 µg/ml CC3 antibody (AF835; R&D Systems) in 1% BSA/PBS or 2.5 µg/ml β‐catenin antibody (610154; BD Transduction Laboratories, Oxford, UK) in PBS was added overnight at 4°C. Bound antibodies were detected with biotinylated goat anti‐rabbit IgG (B7389; Sigma‐Aldrich) or biotinylated goat anti‐mouse IgG (BA9200; Vector Laboratories, Peterborough, UK) and then DyLight‐488 Streptavidin (SA‐5488‐1; Vector Laboratories) diluted 1:200 in PBS. Coverslips were mounted in ProLong Gold and DAPI (P36931; Invitrogen, Paisley, UK).

Brown B.A., Connolly G.M., Mill C.E., Williams H., Angelini G.D., Johnson J.L, & George S.J. (2018). Aging differentially modulates the Wnt pro‐survival signalling pathways in vascular smooth muscle cells. Aging Cell, 18(1), e12844.

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Immunofluorescence Staining of Endothelial Cells

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Cells were fixed with 3% (w/v) paraformaldehyde/PBS for 10 min, then permeabilised with 0.1–1.0% (v/v) Triton X-100/PBS for 15 min. To localize membrane-tethered vWF (excluding intracellular vWF), this permeabilisation step was not performed. Cells were subsequently blocked with 20% (v/v) goat serum/PBS for 30 min, then incubated with primary antibody diluted in PBS, overnight at 4°C. Primary antibodies included: anti-cleaved caspase-3 IgG (R&D Systems; MAB835), anti-NFκB p65 IgG (Cell Signaling Technology; 8242), anti-phospho-paxillin (Tyr118) IgG (Invitrogen; 44-722G), anti-VE-Cadherin IgG (Cell Signaling Technology; 2500), anti-von Willebrand factor IgG (Cell Signaling; 65707S), and anti-ZO-1 IgG (Abcam; ab221547). Cells were then incubated with biotinylated secondary antibody diluted 1:200 in PBS for 30 min, followed by Dylight 488 Streptavidin (Vector Laboratories; SA-5488-1) diluted 1:200 in PBS for 30 min. Prolong Gold Antifade Reagent with DAPI (ThermoFisher Scientific; P36935) was used for staining nuclei and mounting.

Wadey K.S., Somos A., Leyden G., Blythe H., Chan J., Hutchinson L., Poole A., Frankow A., Johnson J.L, & George S.J. (2023). Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction. Frontiers in Cardiovascular Medicine, 9, 1059124.

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Immunohistochemical Detection of PLA2R

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We examined all available serial sections from formalin fixed paraffin blocks (3 micron). Sections were deparaffinized with xylene and treated with proteinase K (20 mg/ml, 15 min at 37°C; Invitrogen) for antigen retrieval. Sections were incubated with rabbit polyclonal anti-PLA₂R1 (1:100; Sigma/Atlas) for 30 min. As we anticipated weak PLA₂R staining in patients with early stages of recurrence or in patients with resolving MN, amplification was used in all cases to optimize staining. The amplification process included sequential blocking with streptavidin and biotin (15 min; Streptavidin/Biotin blocking kit, Vector Laboratories) followed by biotinylated goat anti-rabbit IgG (1:200 for 45 min; Vector Laboratories) and DyLight 488 Streptavidin (1:100 for 45 min; Vector Laboratories).
All staining was done on coded sections without knowledge of the histological or clinical diagnosis and included a known positive control and known negative controls (lupus nephritis or normal human kidney). All glomeruli were photographed at 200x using an Olympus DP72 camera attached to a Nikon epifluorescence microscope. The coded images were scored as positive, negative, or equivocal by five observers, with high inter-observer agreement. Any images on which there was disagreement were reviewed together by all five observers to reach a consensus.

Kattah A., Ayalon R., Beck LH J.r., Sandor D.G., Cosio F.G., Gandhi M.J., Sethi S., Lorenz E.C., Salant D.J, & Fervenza F.C. (2015). Anti-Phospholipase A2 Receptor Antibodies in Recurrent Membranous Nephropathy. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 15(5), 1349-1359.

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Validating SEMA7A Glycosylation in HNSCC

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To confirm glycosylation of the SEMA7A protein, HNSCC cell lines were incubated with tunicamycin and swainsonine (Sigma‒Aldrich, Germany), and cell lysates were incubated with PNGase F and O-glycosidase (New England BioLabs, Ipswich, MA, USA). LCA modification of SEMA7A was further confirmed through lectin blotting, flow cytometry, and immunofluorescence staining by using Biotinylated Lens Culinaris Agglutinin and DyLight 488 Streptavidin (Vector lab).

Liu Z., Meng X., Zhang Y., Sun J., Tang X., Zhang Z., Liu L, & He Y. (2024). FUT8-mediated aberrant N-glycosylation of SEMA7A promotes head and neck squamous cell carcinoma progression. International Journal of Oral Science, 16, 26.

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Biocytin Labeling and Immunostaining

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For analysis of recorded cells, one single RFP⁺ cell per hemisphere was recorded in a slice and loaded with biocytin for 25 min in whole-cell configuration. After gently removing the patch pipette, biocytin was allowed to diffuse for at least 10 min before the slice was fixed 2 hours in 4% paraformaldehyde at 4°C. Then, the slice was rinsed three times in PBS for 10 min and incubated with 1% Triton X-100 and 10% normal goat serum (NGS) for 2 hours. After washing in PBS, slices were immunostained for SOX10, CC1, and STEM101/121. Tissues were incubated with primary antibodies for 3 days at 4°C. Secondary antibodies were diluted in 2% NGS and 0.2% Triton X-100. Tissues were incubated with secondary antibodies for 2 hours at room temperature. Biocytin was revealed with secondary antibodies using DyLight-488 streptavidin (Vector Laboratories, Burlingame, USA, 1:200). Images of biocytin-loaded cells were acquired either with a Carl Zeiss microscope equipped with ApoTome 2 or a LEICA SP8 confocal microscope (63× oil objective; numerical aperture, 1.4; 0.75-μm Z-step) and processed with National Institutes of Health ImageJ software (70 (link)).

Mozafari S., Starost L., Manot-Saillet B., Garcia-Diaz B., Xu Y.K., Roussel D., Levy M.J., Ottoboni L., Kim K.P., Schöler H.R., Kennedy T.E., Antel J.P., Martino G., Angulo M.C., Kuhlmann T, & Evercooren A.B. (2020). Multiple sclerosis iPS-derived oligodendroglia conserve their properties to functionally interact with axons and glia in vivo. Science Advances, 6(49), eabc6983.

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