Texas red avidin

Manufactured by Thermo Fisher Scientific

Sourced in Canada, United States

Texas-Red-avidin is a fluorescent dye-labeled protein used in various biological applications. It is composed of the Texas Red fluorescent dye covalently attached to the avidin protein. Texas-Red-avidin can be used to detect and label biotin-containing molecules, proteins, or cells.

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Texas Red (171 citations) Texas red-conjugated Avidin (2 citations) Alexafluor and Texas red avidin (2 citations)

9 protocols using texas red avidin

Biotinylated AMPA Receptor Labeling in Slices

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Surface labelling of biotinylated AMPARs in slice tissue was performed as described in^{6 (link)}. 20 μΜ biotin (Sigma-Aldrich Cat# B4501) was added to the culture medium after SCE transfection. Four days later, slices were washed in HEPES-based aCSF (see SCE) and excess biotin was removed by dialysis in HEPES-based aCSF against 0.25 ml 2 mg/ml avidin Texas red (Thermo Fisher Scientific Cat# A820) through a Slide-A-Lyzer MINI (3500 molecular weight cut-off, Thermo Fisher) for 45 mins. Slices were then incubated for 45 min at room temperature in HEPES-based aCSF containing 120 nM Streptavidin-AF647 (Invitrogen). Slices were washed three times over 45 mins before fixation in phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 4% sucrose (PFA, Sigma-Aldrich, Cat.# P6148) at 4 °C overnight. Slices were washed and stored in PBS until imaging. For imaging, slices were inverted on a glass-coverslip and imaged in PBS using a 63x/1.4NA oil-immersion objective on a commercial Leica TCS SP8 confocal microscope using LAS X software. Z-stacks of whole cells or whole stratum radiatum dendrites were taken, with EGFP, tdTomato and AF647 excited with 478 nm, 554 nm and 653 nm laser lines, with equivalent power settings across experimental conditions.

Watson J.F., Pinggera A., Ho H, & Greger I.H. (2021). AMPA receptor anchoring at CA1 synapses is determined by N-terminal domain and TARP γ8 interactions. Nature Communications, 12, 5083.

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Lacrimal Gland Tissue Staining

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The lacrimal glands were harvested and formalin-fixed paraffin-embedded (FFPE) sections (4 µm) were prepared and blocked with 2% BSA and anti-FcR antibodies (catalog #14-0161-86, Affymetrix eBioscience)^{48 (link),49 (link)}. Sections were stained with avidin-TexasRed (ThermoFisher) overnight at 4 °C. Slides were then mounted using DAPI-containing VECTASHIELD® mounting medium (Vector Laboratories) and examined under a fluorescence microscope (Nikon Eclipse E800; Nikon Instruments, Melville, NY, USA).

Elbasiony E., Cho W.J., Singh A., Mittal S.K., Zoukhri D, & Chauhan S.K. (2023). Increased activity of lacrimal gland mast cells are associated with corneal epitheliopathy in aged mice. NPJ Aging, 9(1), 2.

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Visualizing TNFα-Induced Membrane Dynamics

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SK-N-AS cells were plated onto 4-compartment glass-bottomed imaging dishes (Greiner Bio-One) in culture medium and incubated at 37 °C in humidified 5% CO₂ on the microscope stage. A Zeiss 780 confocal microscope with a plan-apochromat × 63 NA 1.4 oil objective was used with appropriate excitation and emission signal detection. Image capture was performed using Zeiss software ‘Zen 2010b SP1' to take Z stacks using a stack separation of 0.8–1.2 μM. Maximum intensity projections were used for image analysis. Human recombinant TNFα biotin conjugate (1 μg ml⁻¹, Fluorokine, R&D Systems, Wiesbaden) was diluted to 25 ng ml⁻¹ in either 20 μl of avidin-FITC (10 μg ml⁻¹) or 2 μl avidin-Texas-Red (2 mg ml⁻¹, Life Technologies) and made up to 50 μl with minimum essential medium. Cells were washed with PBS before stimulation. Cells were pretreated with 80 μM Dynasore hydrate (Sigma) for 1 h where applicable. For acid wash treatment, cells were cooled to 4 °C and incubated with acid wash buffer (150 mM NaCl, 100 mM glycine pH 2.5) for 3 × 2 min. Cells were fixed with 3.7% formaldehyde in PBS for 15 min at room temperature, then washed with PBS. Fixed samples were imaged on a Zeiss 780 confocal microscope as above, with a plan-apochromat × 40NA 1.3 oil objective.

Adamson A., Boddington C., Downton P., Rowe W., Bagnall J., Lam C., Maya-Mendoza A., Schmidt L., Harper C.V., Spiller D.G., Rand D.A., Jackson D.A., White M.R, & Paszek P. (2016). Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states. Nature Communications, 7, 12057.

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Avidin-Coated Biotin Microbubbles

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Biotin microbubbles used as “core” bubbles were coated using a molar excess of avidin, from egg (66 kDa), Alfa Aesar (Ward Hill, MA). The avidin solution was prepared by diluting 10 mg of lyophilized avidin in 1 mL of ultrapure water. The solution was rocked for 15 min at room temperature using a benchtop rotoflex tube rotator, Argos Technologies (Vernon Hills, IL), and then stored in a sealed manner at 20 °C. The amount of biotinylated PEG was calculated from the total microbubble area assuming an average lipid headspace and size distribution from the MS4 characterization. Biotinylated microbubbles were added dropwise into a 10-fold molar excess of avidin while the mixture was being mildly vortexed in a 500 μL microcentrifuge tube. The mixture was diluted to 500 μL with PBS in a 1 mL Luer tip syringe and incubated for 15 min with gentle spinning. The incubated solution was then washed three times at 250 RCF for 5 min. After washing, the avidinated microbubbles were characterized using a MS4 coulter counter as described above. Avidin loading on the microbubbles was verified with microscopy and flow cytometry using avidin-Texas Red (68 kDa), Life Technologies (Eugene, OR), in place of the unlabeled avidin.

Hall R.L., Juan-Sing Z.D., Hoyt K, & Sirsi S.R. (2019). Formulation and Characterization of Chemically Cross-linked Microbubble Clusters. Langmuir : the ACS journal of surfaces and colloids, 35(33), 10977-10986.

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GLP1R Internalization Assay in HepG2 Cells

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HepG2 cells were transfected with the hGLP1R-spark-GFP construct (Sino biologics) using lipofectamine-3000 (Thermo Fisher Scientific). Transfected cells were treated with 20 nM of Exendin-4 (Ex-4) (positive control), PK2 (50 μM), PK3 (50 μM), and PK4 (50 μM), and DMSO as a vehicle for 1 h. After completion of incubation, cells were washed with PBS and fixed using formalin. For antagonism of GLP1R receptor, 300 nM of Exendin (9–39) [Ex-9] was pretreated for 15 min followed by compound (PK2-50 μM) treatment as described above. For cell surface labeling, cells were incubated with 1 mg/ml sulpho-NHS-biotin (Thermo Fisher Scientific) in PBS for 30 min at 4 °C, washed with PBS containing 100 mM glycine, and then fixed in 4% PFA for 15 min. Cells were blocked in 1% bovine serum albumin in PBS for another 30 min and then incubated in 5 μg/ml of Texas-Red-avidin (Thermo Fisher Scientific) for 30 min. After extensive washing in PBS, the coverslips were mounted, and internalization of hGLP1R was observed using a Nikon Confocal Microscope.

Girdhar K., Thakur S., Gaur P., Choubey A., Dogra S., Dehury B., Kumar S., Biswas B., Dwivedi D.K., Ghosh S, & Mondal P. (2022). Design, synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor. The Journal of Biological Chemistry, 298(5), 101889.

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Visualizing Nephrocyte Function

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Fluorescent tracer uptake in nephrocytes to evaluate nephrocyte function was performed as previously described.^{39 (link)} Briefly, nephrocytes were dissected in phosphate-buffered saline (PBS) and incubated with fluorescein isothiocyanate (FITC)–albumin (Sigma) or Texas Red–avidin (Thermofisher) for 30 seconds. After a fixation step of 5 minutes in 8% paraformaldehyde, cells were rinsed in PBS and exposed to Hoechst 33342 (1:1000) for 20 seconds and mounted in ROTI-Mount (Carl Roth). Cells were imaged using a Zeiss LSM 880 laser-scanning microscope. Quantitation of fluorescent tracer uptake was performed with ImageJ software. The results are expressed as a ratio to a control experiment with flies carrying the (heterozygous) GAL4 transgene but no UAS that was done in parallel.
For the fluorescent double tracer uptake to study processing of endocytic cargo, nephrocytes were dissected in Schneider’s medium and incubated with FITC-albumin for 30 seconds. Washing steps with cold PBS were followed by a 45-minute chase period in Schneider’s medium at 25 °C and Texas Red–albumin was applied and incubated for 3 minutes. After fixation for 5 minutes in 8% paraformaldehyde, cells were rinsed in PBS and exposed to Hoechst 33342 (1:1000) for 20 seconds and mounted in ROTI-Mount (Carl Roth). Image quantitation was performed using ImageJ software.

Gerstner L., Chen M., Kampf L.L., Milosavljevic J., Lang K., Schneider R., Hildebrandt F., Helmstädter M., Walz G, & Hermle T. (2022). Inhibition of endoplasmic reticulum stress signaling rescues cytotoxicity of human apolipoprotein-L1 risk variants in Drosophila. Kidney international, 101(6), 1216-1231.

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Visualizing Salmon IgM in Fish Spleens

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Three healthy fish were euthanized with an overdose of MS222 (400 mg/L), and their spleens were aseptically collected, homogenized using a 100 µm mesh strainer, and resuspended in FACS media (PBS; 0.1% fetal bovine serum (FBS; Gibco, NY, USA)). Biotinylated chicken IgY anti-salmon IgM (1 mg/mL, Somru BioSciences, Charlottetown, PE, Canada) was labeled with Texas red-avidin (2.5 mg/mL, Thermo Fisher Scientific, MA, USA) by mixing in a 1:1 volume for 30 min at room temperature. Then 50 µL of the spleen cell suspensions were gently mixed with 3 µL of Texas red-IgY anti-salmon IgM, and 1 µL of 4,6-diamidino-2-phenylindole (DAPI, 5 mg/mL concentration) (Thermo Fisher Scientific, MA, USA) in a 1.5 mL centrifuge tube. A 3 µL aliquot of the stained cell suspension was added onto mountain media (Thermo Fisher Scientific, MA, USA) on a microscope slide, covered with a cover slide, and sealed. These samples were visualized by confocal microscope (Nikon AX/AX R, Long Island, NY, USA) for image acquisition. This study was conducted to verify the flow cytometry results.

Ghasemieshkaftaki M., Cao T., Hossain A., Vasquez I, & Santander J. (2024). Haemato-Immunological Response of Immunized Atlantic Salmon (Salmo salar) to Moritella viscosa Challenge and Antigens. Vaccines, 12(1), 70.

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Endothelial Cell Response to Homocysteine Exposure

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HRECs were plated at 1 × 10⁵ in 8-well chamber slides (Sigma-Aldrich Chemical Corp., St. Louis, MO, USA) and treated with and without Hcy (20, 50, or 100 μM) for 24 hr. Cells were then fixed with 4% formalin for 10 min, washed with PBS, and blocked with Power Block (BioGenex, Fremont, CA, Ca. #BS-1310–25) for 1 hr. Thereafter, the cells were incubated at 4 °C overnight with Antibodies for ZO-1(Abcam, Cambridge, Massachusetts, USA, Cat.# ab59720), occludin (Invitrogen, Eugene, Oregon, USA, Mouse monoclonal Cat.# 33–1500), claudin-5 (Invitrogen, Eugene, Oregon, USA, Rabbit Polyclonal Cat.# 34–1600), anti-GSH-1 (Santa Cruz, Dallas, Texas, USA. Cat.# sc-292189), anti-Nrf2 (Abcam, ab137550), anti CD31 (Novus Biologicals, NB100–2284) and anti α-smooth muscle actin (Abcam, ab5694). After primary antibody treatment, cells were then washed 3 times with PBS containing 0.3% Triton-X and incubated with appropriate secondary antibodies (Alexafluor and Texas red avidin, Invitrogen). Slides were cover-slipped using Fluoroshield containing DAPI (Sigma-Aldrich) as a counter stain. Images were captured by fluorescent microscopy (Carl Zeiss, Göttingen, Germany).

Mohamed R., Sharma I., Ibrahim A.S., Saleh H., Elsherbiny N.M., Fulzele S., Elmasry K., Smith S.B., Al-Shabrawey M, & Tawfik A. (2017). Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress. Scientific Reports, 7, 11952.

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Immunofluorescent Analysis of Retinal Cell Metabolism

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Primary mouse cells, ARPE19 cells, and RPE retinal flat-mounts were subjected to immunofluorescent staining to detect GLUT-1 and Glycolytic enzymes (HK-1 and LDH). First, ARPE19 and primary RPE cells were fixed in 4% paraformaldehyde for 10 min then washed with PBS and blocked with Power Block (BioGenex, Fremont, CA, USA, Cat. # BS-1310-25) for one hour. Then cells were incubated with anti-HK-1 (Abcam, MA, USA, Cat. # ab82832), anti-GLUT-1 (Cell Signaling, Danvers, MA, USA. Cat. # 5704S), and anti-LDH (Thermofisher, MA, USA, Cat. #5-13182) for three h at 37 °C. Then, cells were washed with PBS containing 0.3% Triton-X 3 times. After washing, cells were incubated with appropriate secondary antibodies (Alexa fluor and Texas red avidin, Invitrogen, Eugene, Oregon) and coverslipped with DAPI (Sigma-Aldrich Chemical Corp., St. Louis, MO, USA). Images were captured by a fluorescent microscope (Carl Zeiss, Göttingen, Germany) with a high-resolution camera using Zeiss Axiovision digital image processing software (version 4.8). GLUT-1 expression was evaluated in RPE flat-mounts isolated from mice injected intravitreally with Hcy (WT and NMDARR^−/− mice). RPE flat-mounts were conducted per our published methods [11 (link),15 (link)].

Samra Y.A., Zaidi Y., Rajpurohit P., Raghavan R., Cai L., Kaddour-Djebbar I, & Tawfik A. (2023). Warburg Effect as a Novel Mechanism for Homocysteine-Induced Features of Age-Related Macular Degeneration. International Journal of Molecular Sciences, 24(2), 1071.

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