MO3.13 cells were plated on glass cover slips (25000 cells/cm2) into 24-well plates for 2 days and then treated with serum-free medium. After 24 h of treatment, cells were fixed for 30 min in 2% paraformaldehyde, permeabilised with 0.1% Tween-20 and blocked with 5% horse serum for 15 min. Cells were then incubated overnight with anti-myelin basic protein antibody (1 : 50, AbCam ab62631). Cover slips were washed with PBS (10 min for three times), incubated with anti-rabbit biotinylated secondary antibody (Jackson Immuno Research Europe, Suffolk, UK, 1 : 500) for 90 min, washed with PBS and then stained with Alexa 488 Streptavidin (Molecular Probes, Netherlands, 1 : 1000) for 30 min. Finally, the cover slips were mounted onto glass microscope slides in presence of Vectashield mounting medium with DAPI (Vector Laboratories inc. Burlingame, CA, USA). Confocal images were acquired as z-stacks by using a scanned on z axis with a SP2 TCS-NT Leica (Nussloch, Germany) laser scanning confocal microscope, equipped with 40 × 1.2 NA Plan Apo oil objective. The optical slice was set to 1 μM (FWHM).
Streptavidin alexa 488
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Netherlands, Germany
Streptavidin-Alexa 488 is a fluorescently labeled streptavidin conjugate. Streptavidin is a protein that binds strongly to the vitamin biotin. The Alexa Fluor 488 dye is covalently attached to the streptavidin, providing a fluorescent label.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Streptavidin alexa 594, by Thermo Fisher Scientific (3 mentions)
Ix81 fluorescence microscope, by Olympus (3 mentions)
Hoechst 33258, by Thermo Fisher Scientific (3 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (2 mentions)
Alexa 594 labeled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Ez link sulfo nhs lc biotin, by Thermo Fisher Scientific (2 mentions)
Biotinylated lectin, by Vector Laboratories (2 mentions)
Isolectin b4, by Vector Laboratories (2 mentions)
Anti ki67, by Abcam (2 mentions)
Anti giantin antibody, by Abcam (2 mentions)
Biotinylated vvl, by Vector Laboratories (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Anti digoxigenin rhodopsin, by Thermo Fisher Scientific (2 mentions)
Biocytin, by Merck Group (2 mentions)
Alexa 647, by Thermo Fisher Scientific (2 mentions)
Donkey anti rabbit 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 594 goat anti rat igm, by Thermo Fisher Scientific (2 mentions)
88 protocols using streptavidin alexa 488
1
Immunocytochemistry of Myelin Basic Protein
2
Lectin-Based Quantification of Striatal Microvessels
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Striatal sections from vehicle- and GA-injected animals at 14 and 30 DPI were permeabilized with 1% Triton X-100 for 20 min at RT, quickly rinsed and incubated overnight with 5 μg/ml of Lycopersicon esculentum biotinylated lectin (Sigma). After 3 washes with PBS, sections were incubated with a 1:500 dilution of Alexa 488 streptavidin (Molecular Probes®). After 90 min, sections were washed 3 times, mounted in 50% glycerol and imaged in a confocal microscope. Green positive areas of blood vessels were quantified for both experimental conditions after manually delineating all vessel profiles <10 μm in diameter in a field of 211.5 mm2. At least 5–7 fields per section, 3–5 sections per animal and 3–5 animals per condition were analyzed.
3
Amoxicillin Reactivity and Protein Labeling
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Amoxicillin was from Glaxo Smithkline Beecham (Madrid, Spain). Deuterium oxide for NMR studies (D2O) was from Panreac Química SAU (Barcelona, Spain). Potassium phosphate buffer (PBS) for 1H-NMR reactivity studies was prepared in 99.8% D2O at pH 7.4. HRP-Streptavidin and ECL system were from GE Healthcare. Alexa-488 streptavidin was from Molecular Probes. Albumin from human serum, phorbol myristate acetate, iodoacetamide and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) were from Sigma. Biotin-NHS ester was from Aldrich.
4
Neurobiotinic Trace of Moth Olfactory System
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Six male and six female moths were anesthetized with carbon dioxide and immobilized using double-sided adhesive tape, and the ventral part of the head was exposed. The antennae or the palp of one side of the moth were cut near the base, and the remainder of the segments were inserted for 30 min in an appropriately sized micropipette tip filled with 2% Neurobiotin (Neurobiotin Tracer, Vector Laboratories, Burlingame, USA) solution. After the micropipette was removed, the preparations were then incubated at 4°C for 2 h in a humidified and dark box for diffusion of the dye. Subsequently, the brains were dissected out in 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.2) under a dissecting microscope, and the isolated brains were fixed in the same fixative at 4°C for 1-2 days for batch processing. After rinsing three times with 0.1% Triton X-100 in 0.1 M PB, the brains were incubated in 2 μg/ml Alexa-488 Streptavidin (Molecular Probes, Life Technologies, USA) solution in PBS for 1-2 days and then in 10 μg/ml Propidium Iodide (Sigma) solution for 30 min at room temperature. The brains were dehydrated in an ascending ethanol series of 30, 50, 75, 95, and 100% alcohol and then embedded in DPX (Sigma) after a brief xylene transition.
5
Hyaluronan Staining of Unstimulated CD4+ T Cells
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We adapted a method from Lin37 (link) to stain HA on the unstimulated CD4+ T cell surface. Unstimulated CD4+ T cells were fixed by acid–formalin–ethanol and stained with biotinylated hyaluronan-binding protein (Calbiochem, Gibbstown, NJ, USA) overnight, then stained with Alexa488-streptavidin (Life Technologies) for 2 h. HA staining was measured by two methods: laser scanning microscopy and flow cytometry (fluorescence-activated cell sorting). For laser scanning microscopy analysis, the cells were further stained, cell membranes were stained with Texas Red (Life Technologies) and nuclei stained with DAPI (4,6-diamidino-2-phenylindole; Life Technologies). LSM510 META laser scanning microscope (Carl Zeiss Microimaging Inc., Thornwood, NY, USA) was used to scan the cells with Z-stack projections. For fluorescence-activated cell sorting analysis, 50 000 HA-stained cells were analyzed by EasyCyte6HT-2L (Millipore, Billercia, MA, USA).
6
Immunofluorescent Staining of Angiogenesis
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Recombinant human VEGF was purchased from R&D Systems (Minneapolis, MN, www.rndsystems.com ). Anti-CD31 antibody (MEC 13.3) and growth factor reduced Matrigel were purchased from BD Biosciences (Bedford, MA). Anti-PCNA antibody (FL-261) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Rabbit anti-α-SMA antibody (ab5694) was purchased from Abcam (Cambridge, United Kingdom). Biotinylated-ILB4 was purchased from Vector Laboratories (Burlingame, CA). Alexa 488 Streptavidin, Alexa 488 goat anti-rabbit, Alexa 488, 568 and Alexa 647 goat anti-rat secondary antibodies were purchased from Life Technologies (Carlsbad, CA). Ac-PGP (Ac-Pro-Gly-Pro-OH, purity >95%) was purchased from Anaspec Inc. (Fremont, CA).
7
Evaluating Tight Junction Permeability in Mice
8
Evaluating Tight Junction Permeability in Mice
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To test the TJ barrier permeability in mice, 10 mg/ml EZ-Link sulfo-NHS-LC-biotin (Pierce Chemical) in PBS containing 1 mM CaCl2was intradermally injected underneath the dermis of the E18.5 mice back skin. After 30 minutes of incubation at 37°C, mice back skin was isolated, embedded in Tissue-Tek, and frozen. 4% Paraformaldehyde fixed section was immunostained with TJ marker, anti-Occludin antibody. After washing step, Alexa 594labeled secondary antibodies (Life technologies)for TJ marker and Alexa 488 Streptavidin (Life technologies)for biotin labeling were applied for 30 minutes at RT. Followed by another washing and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen).Image were taken using an Olympus BX43.
9
Detecting Cell Signaling Pathways in CNV Model
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Antibodies used for this study include rabbit antibodies against VEGFR2 (Cell Signaling Technology, Danvers, MA, USA) and GAD65 (Abcam, Cambridge, MA, USA), goat antibody against Centaurin A (Santa Cruz Biotechnology, Dallas, TX, USA), mouse antibodies against rhodopsin (Millipore, Burlington, MA, USA) and glutamine synthetase (BD Bioscience, Franklin Lakes, NJ, USA). To neutralize VEGF in the mouse laser-induced CNV model, a mouse-specific antibody against VEGF (512807; BioLegend, San Diego, CA, USA) and the IgG control (400515; BioLegend) was purchased from the same company. Aflibercept was obtained from the eye clinic adjacent to the Department of Ophthalmology at UIC. Other reagents used were isolectin B4 (Vector Laboratories, Burlingame, CA, USA), Alexa 594 anti-rabbit antibody, Alexa 488 anti-mouse antibody, Alexa 488-streptavidin (Invitrogen, Carlsbad, CA, USA), and HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA, USA).
10
Immunofluorescence Staining of Pdot-labeled MSCs
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Immunofluorescence staining of the Pdot-labeled MSCs was performed in accordance of manufacturers' instructions. In brief, cells were fixed with 4% PFA for 20 mins at room temperature, permeabilized by using 0.5% TtitonX-100 for 20 mins at room temperature. After blocking with 5% BSA for 1 h at 37 °C, cells were incubated with primary antibodies including OCT-4 (ab19857, Abcam), NANOG (ab109250, Abcam), and SSEA-4 (ab16287, Abcam) at 4 °C overnight, followed by incubation with Alexa Fluor® 488-labeled secondary antibodies for 2 hours at room temperature in dark. Tubulin structure was stained by biotin anti-α-tubulin antibody (627904, Biolegend) and Alexa 488-streptavidin (S11223, Invitrogen).
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