Nucblue fixed cell stain readyprobe

Manufactured by Thermo Fisher Scientific

Sourced in United States

NucBlue Fixed Cell Stain ReadyProbes is a lab equipment product that stains the nuclei of fixed cells. It is a ready-to-use solution that can be directly applied to samples.

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Lab products found in correlation

Inverted microscope, by Zeiss (10 mentions) Apotome, by Zeiss (8 mentions) Triton x 100, by MP Biomedicals (3 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions) Actinred 555 readyprobe, by Thermo Fisher Scientific (2 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions) Doxycycline, by Merck Group (2 mentions) Matrigel, by Corning (2 mentions) Lsm 510 meta confocal microscope, by Zeiss (2 mentions) Lsm 780, by Zeiss (2 mentions) Alexa fluor 555 phalloidin, by Cell Signaling Technology (2 mentions) Integrin α2, by Abcam (1 mentions) F actin, by Thermo Fisher Scientific (1 mentions) Rab11, by Thermo Fisher Scientific (1 mentions) Proteostat, by Enzo Life Sciences (1 mentions) E cadherin, by Thermo Fisher Scientific (1 mentions) Nak atpase, by Thermo Fisher Scientific (1 mentions) Hsp90, by Cell Signaling Technology (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions)

16 protocols using nucblue fixed cell stain readyprobe

Cardiac Troponin T Immunostaining in AC16 Cells

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On day 1, 3, and 5, AC16 samples were rinsed with PBS (Gibco) and fixed for 10 minutes with 4% paraformaldehyde. PFA was removed and samples rinsed using PBS twice. Samples were permeabilized for 1 minute with 0.1% Triton, rinsed two times with PBS, and blocked using 2% BSA (Bovine Serum Albumin, Fisher Bioreagents) for 30 minutes at 4°C. Samples were stained with 1:200 cardiac troponin T monoclonal (13 – 11 antibody, ThermoFisher) in PBS overnight at 4°C. Samples rinsed twice with PBS and stained with Alexa Fluor 488 goat anti-mouse secondary antibody (1:200) and Actin-stain 555 phalloidin (ActinRed 555 ReadyProbes, Invitrogen) in PBS at 1 hour at RT. Samples were rinsed twice with PBS. Right before imaging, sample nuclei were stained with DAPI (NucBlue Fixed Cell Stain ReadyProbes, Invitrogen) for 10 minutes at RT and rinsed with PBS once. Fluorescent images were captured with a Leica Thunder Imager Live Cell and 3D Assay fluorescent microscope with a 10x objective (Leica Microsystems, Buffalo Grove, IL).

Esparza A., Jimenez N., Joddar B, & Natividad-Diaz S. (2023). Development of in vitro cardiovascular tissue models within capillary circuit microfluidic devices fabricated with 3D Stereolithography printing. Research Square.

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Organoid Differentiation and Imaging Protocol

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Organoids were seeded in 3D in Matrigel (Corning) plugs as for passaging organoids. Following seeding, organoids were given hW-CMGF+ medium^{44 (link)} supplemented with 10 µmol Y-27632 Rock inhibitor, and for J2-NGN3 organoids, 200 µg/ml Geneticin for 2 days. After two days, media were changed to according to organoid treatment group: undifferentiated: hW-CMGF+ medium, 10 µmol Y-27632 Rock inhibitor, and for J2-NGN3 organoids 200 µg/ml Geneticin; differentiated and uninduced: differentiation medium^{44 (link)} with 10 µmol Y-27632 Rock inhibitor; differentiated and induced (J2-NGN3 organoids only): differentiation medium, 10 µmol Y-27632 Rock inhibitor, and 1.0 µg/ml doxycycline (as doxycycline monohydrate, Sigma-Aldrich, St. Louis, MO, USA, dissolved in DMSO). These media conditions (at 500 µl) were continued for 4 days with daily media changes. After 4 days, 3D organoids were prepared for imaging^{79 (link)}. Epithelial cell boundaries were imaged by staining with Alexa 647 conjugated E-cadherin, and nuclei were stained with 0.07x NucBlue Fixed Cell Stain ReadyProbes (Invitrogen, USA) with secondary antibody application. Primary and secondary antibodies and their dilutions are listed in Supplementary Table 8.

Danhof H.A., Lee J., Thapa A., Britton R.A, & Di Rienzi S.C. (2023). Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling. bioRxiv.

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Cellular Internalization of Labeled Polymers

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ATDC5 cells (20,000) were seeded onto an 8-well chambered coverglass (#1 German borosilicate, Nunc LabTek) and allowed to adhere overnight. Cells were washed with PBS and then treated for 24 h with 250 μg/mL Cy5-labeled polymers dissolved in DMEM + 1% FBS + 1% P/S. After incubation, cells were washed with PBS, fixed with 4% paraformaldehyde in PBS (Alfa Aesar) for 25 min, washed again with PBS, stained with DAPI (NucBlue Fixed Cell Stain ReadyProbes, Invitrogen) based upon manufacturer directions, washed again with PBS, and overlaid with 25 mM tris(2-carboxyethyl)phosphine-supplemented PBS to reduce extracellular fluorescence of Cy5 as previously described.^{80 (link)} Finally, cells were imaged with a confocal scanning laser microscope (Nikon Eclipse Ti Microscope with D-Eclipse C1 laser, Nikon Instruments, Inc.) using a 405 nm (blue) and 640 nm (red) laser line. Resulting images were processed by splitting the blue and red channels and merging images.

DeJulius C.R., Dollinger B.R., Kavanaugh T.E., Dailing E., Yu F., Gulati S., Miskalis A., Zhang C., Uddin J., Dikalov S, & Duvall C.L. (2021). Optimizing an Antioxidant TEMPO Copolymer for Reactive Oxygen Species Scavenging and Anti-Inflammatory Effects in Vivo. Bioconjugate chemistry, 32(5), 928-941.

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Organoid Differentiation and Imaging

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Organoids were seeded in 3D in Matrigel (Corning, NY, USA) plugs as for passaging organoids. Following seeding, organoids were given hW-CMGF+ medium^{42 (link)} supplemented with 10 µmol Y-27632 Rock inhibitor, and for J2-NGN3 organoids, 200 µg/ml Geneticin for 2 days. After two days, media were changed according to organoid treatment group: undifferentiated: hW-CMGF+ medium, 10 µmol Y-27632 Rock inhibitor, and for J2-NGN3 organoids 200 µg/ml Geneticin; differentiated and uninduced: differentiation medium^{42 (link)} with 10 µmol Y-27632 Rock inhibitor; differentiated and induced (J2-NGN3 organoids only): differentiation medium, 10 µmol Y-27632 Rock inhibitor, and 1.0 µg/ml doxycycline (as doxycycline monohydrate, Sigma-Aldrich, St. Louis, MO, USA, dissolved in DMSO). These media conditions (at 500 µl) were continued for 4 days with daily media changes. After 4 days, 3D organoids were prepared for imaging using established methods.^{79 (link)} Epithelial cell boundaries were imaged by staining with Alexa 647 conjugated E-cadherin, and nuclei were stained with 0.07× NucBlue Fixed Cell Stain ReadyProbes (Invitrogen, USA) with secondary antibody application. Primary and secondary antibodies and their dilutions are listed in Supplemental Table S8.

Danhof H.A., Lee J., Thapa A., Britton R.A, & Di Rienzi S.C. (2023). Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling. Gut Microbes, 15(2), 2256043.

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Comprehensive Immunofluorescence Assay Protocols

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For immunofluorescence, antibodies and the probes used were as follows: F-actin (phalloidin–Alexa Fluor 488, catalog A12379 or phalloidin–Alexa Fluor 647, catalog A22287, Invitrogen); nuclei (NucBlue Fixed Cell Stain Ready Probes, catalog R37606, Invitrogen); aggresome (ProteoStat, catalog ENZ-51038, Enzo Life Sciences Inc.); UNC45A (catalog sc-101493, 1:50, Santa Cruz Biotechnology Inc.); villin (catalog sc-58897, 1:50, Santa Cruz Biotechnology Inc.); DRA (catalog ab83545, 1:100, Abcam); Rab 11 (catalog 71-5300, 1:25, Invitrogen); NHE3 (catalog NBP-82574, 1:500, Novus Biologicals); MYO5B (catalog HPA069773, 1:400, Sigma-Aldrich); Epcam (catalog AF960, 1:100, R&D); CFTR (catalog ab59394, 1:100, abcam); NA/K-ATPase (catalog MA5-32184, 1:100, Thermo Fisher); integrin-α2 (catalog ab181548, 1:100, Abcam); and E-cadherin (catalog 13-700445, 1:100, Thermo Fisher). For STED, F-actin (phalloidin, phalloidin-ATTO 550, Invitrogen) was used. For Western blot, the following antibodies were used: actin (catalog 5125, 1:1000, Cell Signaling); FLAG (catalog F3165, 1:1000, Sigma-Aldrich); GAPDH (catalog 14C10, 1:1000, Cell Signaling Technology); HSP90 (catalog 4874, 1:1000, Cell Signaling Technology); UNC45A (catalog 171328, 1:500, Abcam); and MYO5B (catalog PA5-49519, 1:500, Thermo Fisher).

Duclaux-Loras R., Lebreton C., Berthelet J., Charbit-Henrion F., Nicolle O., Revenu des Courtils C., Waich S., Valovka T., Khiat A., Rabant M., Racine C., Guerrera I.C., Baptista J., Mahe M.M., Hess M.W., Durel B., Lefort N., Banal C., Parisot M., Talbotec C., Lacaille F., Ecochard-Dugelay E., Demir A.M., Vogel G.F., Faivre L., Rodrigues A., Fowler D., Janecke A.R., Müller T., Huber L.A., Rodrigues-Lima F., Ruemmele F.M., Uhlig H.H., Del Bene F., Michaux G., Cerf-Bensussan N, & Parlato M. (2022). UNC45A deficiency causes microvillus inclusion disease–like phenotype by impairing myosin VB–dependent apical trafficking. The Journal of Clinical Investigation, 132(10), e154997.

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Immunofluorescence Staining of HUVEC Cells

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On day 1, 3 and 5, HUVEC samples were rinsed with PBS (Gibco) and fixed with 4% paraformaldehyde (PFA, Paraformaldehyde Solution in 4% PBS, Thermo Scientific) for 10 minutes. PFA was removed and rinsed with PBS twice. Samples were permeabilized for 1 minute with 0.1% Triton, rinsed two times with PBS, and blocked with 2% BSA (Bovine Serum Albumin, Fisher Bioreagents) for 30 minutes at 4°C. Samples were stained with 1:200 primary anti-human CD31 (CD31 Monoclonal Antibody WM59, Invitrogen) in PBS overnight at 4°C. The samples were rinsed twice PBS and stained with Alexa Fluor 488 goat anti-mouse secondary antibody (1:200) and Actin-stain 555 phalloidin (ActinRed 555 ReadyProbes, Invitrogen) in PBS for 1 hour at room temperature (RT). Samples were rinsed twice with PBS. Right before imaging, sample nuclei were stained with DAPI (NucBlue Fixed Cell Stain ReadyProbes, Invitrogen) for 10 minutes at RT and washed with PBS once. Fluorescent images were captured with a Leica Thunder Imager Live Cell and 3D Assay fluorescent microscope with a 10x objective lens (Leica Microsystems, Buffalo Grove, IL).

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GPC3 Antibody Immunofluorescence and Flow Cytometry

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A GPC3 antibody conjugated to the phycoerythrin (PE) fluorophore (clone 1G12 + GPC3/863, NBP2-47763PE, Novus Biologicals) was used with immunofluorescence and flow cytometry experiments as described at a dilution of 1:100. For living cells, DAPI (cat. no. R37605, NucBlue Live Cell Stain ReadyProbes, Invitrogen) was used according to the manufacturer’s protocol for microscopy experiments. For fixed cells, DAPI (cat. no. R37606, NucBlue Fixed Cell Stain ReadyProbes, Invitrogen) was used for flow cytometry experiments at a dilution of 2 drops per 1 mL.

Espinoza A.F., Kureti P., Patel R.H., Do S.L., Govindu S.R., Armbruster B.W., Urbicain M., Patel K.R., Lopez-Terrada D., Vasudevan S.A, & Woodfield S.E. (2024). An indocyanine green-based liquid biopsy test for circulating tumor cells for pediatric liver cancer. Hepatology Communications, 8(6), e0435.

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Imaging DNA Damage Response Proteins

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Cells were seeded (2×10⁵ cells per dish) in 35 mm glass bottomed petri dishes containing an etched grid (MatTek, Ashland, MA) and incubated in medium supplemented with 10 µM BrdU. After 24 h, DNA single strand breaks and base lesions were introduced by low power UV laser micro-irradiation at 364 nm [7 (link)]. Cells were imaged using a 40× C-Apochromat (numerical aperture 1.2) water immersion objective coupled to a Zeiss LSM510 META confocal microscope (Carl Zeiss MicroImaging).
Cells were fixed in 4% paraformaldehyde 1 min after UV damage, permeabilized with 0.25% Triton X-100 in PBS for 10 min, washed three times in PBS, then further permeabilized and blocked with PBS + 1% BSA for 30 min. Following incubation with anti- XRCC1 antibody (1:50; Abcam ab1838) and anti-PADPR antibody (1:100; Abcam ab14460) for 1 h, cells were washed three times with PBS, then incubated with Alexa 488 conjugated anti-mouse and Alexa 647 conjugated anti-chicken secondary antibodies (1:2,000; Life Technologies) for 1 h. Cells were then washed three times with PBS, and the nucleus was stained with NucBlue® Fixed Cell Stain ReadyProbes™ (Life Technologies) for 5 min. Fluorescence images were acquired with the same 40× water immersion objective on the LSM510. Recruitment of XRCC1 and accumulation of PAR at the site of DNA damage were measured using IMAGEJ as described previously [7 (link)].

Horton J.K., Gassman N.R., Dunigan B.B., Stefanick D.F, & Wilson S.H. (2014). DNA polymerase β-dependent cell survival independent of XRCC1 expression. DNA repair, 26, 23-29.

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Imaging and Quantification of Cell Adhesion

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HUVECs or HUASMCs were seeded onto protein-coated or non-coated surfaces of 96-well borosilicate cell imaging plates at a density of 1.0 × 10⁴ cells/well with 100 µl cell culture medium. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂. After 24 h, wells were rinsed with PBS to remove unattached cells. Samples were then fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Japan), washed with PBS and permeabilized with 0.5% Triton-X-100 (Wako Pure Chemical Industries, Japan) for 25 min. Nonspecific binding sites were blocked with 1% bovine serum albumin (BSA, Wako Pure Chemical Industries, Japan) for 1 h. Thereafter, cells were incubated overnight at 4°C with rabbit monoclonal anti-human vinculin antibody (Life Technologies, USA) in 1% BSA. Cells were stained with Alexa Fluor™ 546 goat anti-rabbit IgG (Life Technologies, USA) in 1% BSA for 1 h at room temperature, Alexa Fluor™ 488 phalloidin (Life Technologies, USA) in PBS for 30 min at 4°C and NucBlue™ Fixed Cell Stain ReadyProbes™ (Life Technologies, USA) in PBS for 5 min at room temperature. Fluorescent images were acquired using a fluorescence microscope (BZ-X710, KEYENCE, Japan). All experiments were performed in hexaplicate. Cell numbers and coverage were calculated using built-in Hybrid Cell Count software from 20 or more images.

Natsume K., Nakamura J., Sato K., Ohtsuki C, & Sugawara-Narutaki A. (2022). Biological properties of self-assembled nanofibers of elastin-like block polypeptides for tissue-engineered vascular grafts: platelet inhibition, endothelial cell activation and smooth muscle cell maintenance. Regenerative Biomaterials, 10, rbac111.

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Multifaceted Cellular Imaging Protocol

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Following treatment, cells were fixed with 4% paraformaldehyde (PFA; 15 min), quenched with 0.1 M glycine-PBS (5 min) and permeablised using 0.1% Triton-X-100 (5 min). Cells were then incubated with the following primary antibodies: anti-Calreticulin (Cell Signalling, UK; #12238), anti-YAP1 (Abcam, UK; ab56701 or ab52771), anti-8OHdG (Abcam, UK; ab62623), anti-Hoechst 33342 (NucBlue^® fixed cell stain ready probes™, Life Technologies, UK; R37606). Secondary antibodies Alexa fluor^® 488 goat anti-mouse IgG (H + L) and Alexa fluor® 488 goat anti-rabbit IgG (H + L) (Thermo Fisher Scientific, UK; A-11001 and A-11034 respectively) and DyLight 649 horse anti-mouse IgG (H + L) and DyLight 649 horse anti-rabbit IgG (H + L) (Vector Laboratories, UK; DI-2649 and DI-1649 respectively) were used. Cells were imaged on a ZEISS LSM 780 confocal microscope (ZEISS Microscopy, UK) using the Plan-Apochromat 100x/1.4 Oil DIC objective (1024 × 1024 pixels; 8-bit. Pinhole 100 µm). Pseudo-colouring was applied to the images using Zen 2012 SP1 software (ZEISS; black edition, version 8.1): UV channel (blue), 488 channel (green) and 633 channel (red).

Eales K.L., Wilkinson E.A., Cruickshank G., Tucker J.H, & Tennant D.A. (2018). Verteporfin selectively kills hypoxic glioma cells through iron-binding and increased production of reactive oxygen species. Scientific Reports, 8, 14358.

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