T2767

Manufactured by Thermo Fisher Scientific

Sourced in United States

The T2767 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. The core function of the T2767 is to provide a reliable and precise solution for laboratory tasks.

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Goat anti-rabbit Texas-red (T2767)(

8 protocols using t2767

Immunostaining for HIF-1α and LC3B

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Immunostaining protocol was used as comprehensively described elsewhere [54 (link)]. Briefly, fixed cells were incubated with primary antibodies anti-HIF-1α (ab51608, 1:500) and anti-LC3B (ab52862, 1:500) (Abcam, Cambridge, UK) at 4°C overnight and secondary antibody conjugated to Texas Red (1:1000, T2767) (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. Nuclei were visualized using DAPI staining and F-actin was detected using phalloidin staining. Digital cell images were captured using an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software.

Lewinska A., Adamczyk-Grochala J., Bloniarz D., Horeczy B., Zurek S., Kurowicki A., Woloszczuk-Gebicka B., Widenka K, & Wnuk M. (2020). Remifentanil preconditioning protects against hypoxia-induced senescence and necroptosis in human cardiac myocytes in vitro. Aging (Albany NY), 12(14), 13924-13938.

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Immunofluorescence Analysis of Stress Granule Proteins

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Cells seeded in a 48-well CytoOne tissue culture-treated plate (USA Scientific) were treated with arsenite, fixed in 2% paraformaldehyde for 15 min. at room temperature, and washed twice with PBS for 5 min. The following steps were performed at room temperature unless specified otherwise. Cells were permeabilized with 0.2% Triton X-100 (in PBS) for 10 min and washed twice with PBS for 5 min. To limit nonspecific antibody binding, cells were blocked with 5% bovine serum albumin (BSA) (in PBS) overnight at 4^oC. Cells were incubated for two hours with primary antibodies specific for G3BP1 (1:200, Proteintech, 66486–1-Ig or 13057–2-AP), ZBP1 (1:200, Novus Biologicals, NBP1–76854), TIAR (1:100, Santa Cruz Biotechnology, sc-398372), and RIPK3 (1:50, ProScience, 2293) diluted in 2% BSA (in PBS) as indicated and washed twice with PBS for 10 min. Cells were incubated for 1 hour in the dark with secondary antibodies (1:500, Thermo Fisher, T862, T2767, A11001 or 1:1000, ThermoFisher, A32731) diluted in 2% BSA (in PBS) as indicated and washed twice with PBS for 10 min. Cells were stained with DAPI (4′,6-diamidino-2-phenylindole) (in PBS) for 15 min and washed twice with PBS for 10 min. Cells were imaged and quantified using EVOS FL auto imaging system (Invitrogen).

Szczerba M., Johnson B., Acciai F., Gogerty C., McCaughan M., Williams J., Kibler K.V, & Jacobs B.L. (2023). Canonical cellular stress granules are required for arsenite-induced necroptosis mediated by Z-DNA-binding protein 1. Science signaling, 16(776), eabq0837.

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Immunofluorescence Staining of HT29 Cells

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HT29 cells were fixed with 2% paraformaldehyde for 20 min and permeabilized with 0.1% Triton-X100 for 10 min. After that, HT29 cells were sequentially incubated with blocking solution (PBS with 10% serum and 1% BSA) at room temperature for 1 h, with primary antibodies anti-Vimentin (1:100, Abcam ab92547) or anti-E-Cadherin (1:100, ThermoFisher RA222618) at 4 °C overnight, and a secondary antibody (anti-mouse-FITC, 1:200, Invitrogen F2761 for E-Cadherin staining, or anti-rabbit-TexasRed, 1:200, ThermoFisher T2767 for Vimentin staining) for 45 min at room temperature. All nuclei were stained with Hoechst33342 (2 µM). Cells and intestinal grafts were visualized with the confocal microscope Leica TCS SP8, and pictures were taken with the software LASX (Leica Application Suite X).

Ortiz-Masiá D., Gisbert-Ferrándiz L., Bauset C., Coll S., Mamie C., Scharl M., Esplugues J.V., Alós R., Navarro F., Cosín-Roger J., Barrachina M.D, & Calatayud S. (2020). Succinate Activates EMT in Intestinal Epithelial Cells through SUCNR1: A Novel Protagonist in Fistula Development. Cells, 9(5), 1104.

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Senescent Breast Cancer Cells: Polyphenol Effects

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Doxorubicin-induced senescent breast cancer cells were treated with 0.62% encapsulated polyphenolic extracts (without fluorescein) for 24 h, fixed, and the levels of cell cycle inhibitor p21, pro-inflammatory cytokine IL8, and autophagic markers beclin-1 and LC3b were then evaluated using dedicated antibodies, namely primary antibodies anti-p21 (1:800, MA5-14949), anti-IL8 (1:500, ab154390), anti-BECN1 (1:100, TA502527), and anti-LC3b (1:500, PA5-32254), and secondary antibodies conjugated with fluorochromes (anti-rabbit secondary antibody conjugated with Texas Red (1:1000, T2767), anti-rabbit secondary antibody conjugated with FITC (1:1000, T2765), and anti-mouse secondary antibody conjugated with cyanine 5 (1:1000, A10524) (Thermo Fisher Scientific, Waltham, MA, USA, Abcam, Cambridge, UK)). Furthermore, using encapsulated polyphenolic extract containing fluorescein, microcapsule uptake was also tracked as increased intracellular fluorescence signals. Fluorescence signals were analyzed using a confocal imaging system IN Cell Analyzer 6500 HS and IN Carta software 1.14 (Cytiva, Marlborough, MA, USA). The uptake of microcapsules and the levels of p21, IL8, beclin-1, and LC3b are presented in relative fluorescence units (RFU).

Hajri L., Lewińska A., Rzeszutek I., Oklejewicz B., Wojnarowska-Nowak R., Krogul-Sobczak A., Szpyrka E., Aires A., Ghodbane S., Ammari M, & Wnuk M. (2024). Anticancer Activity of Encapsulated Pearl Millet Polyphenol-Rich Extract against Proliferating and Non-Proliferating Breast Cancer Cells In Vitro. Cancers, 16(9), 1750.

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Quantification of Cellular Biomarkers

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An immunostaining procedure was used as described comprehensively elsewhere [21] (link). The following primary and secondary antibodies were used: anti-Ki67 (1:500, PA5-19462), anti-53BP1 (1:100, PA5-17578) (Thermo Fisher Scientific) and anti-DNMT2 (1:200, sc-365001, ab82659, PA5-11187) and a secondary antibody conjugated to Texas Red (1:1000, T2767) (Thermo Fisher Scientific). Digital cell images were captured using imaging cytometry (In Cell Analyzer 2000 equipped with a high performance CCD camera, GE Healthcare, Little Chalfont, UK). Ki67 signals were scored [%], 53BP1 foci were scored per nucleus and DNMT2 cytosolic signals were presented as relative fluorescence units (RFU). Moreover, DNMT2 nuclear/cytosolic ratio was calculated.

Lewinska A., Adamczyk-Grochala J., Kwasniewicz E., Deregowska A., Semik E., Zabek T, & Wnuk M. (2017). Reduced levels of methyltransferase DNMT2 sensitize human fibroblasts to oxidative stress and DNA damage that is accompanied by changes in proliferation-related miRNA expression. Redox Biology, 14, 20-34.

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Autophagy Induction in Mtb-Infected Macrophages

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Naive MΦ were infected using GFP expressing Mtb (MOI=1) for 4 h, washed and treated with either CD44TA-LIP or left untreated and incubated until 18 h post infection. The cells were then washed, plated onto coverslips, fixed in 3.7% paraformaldehyde, permeabilized using tween80-digitonin--BSA buffer. A specific mAb to LC3 (Cell signaling #3868) was used to stain autophagosomes using a Texas red conjugated secondary antibody (ThermoFisher Scientific, T-2767). IF images were acquired using a NIS element deconvolution software and N90 Nikon microscope. Colocalization was determined by scoring the localization of LC3B and GFP Mtb phagosomes of 25 MΦ in triplicate chambers per treatment group.

Singh V.K., Chau E., Mishra A., DeAnda A., Hegde V.L., Sastry J.K., Haviland D., Jagannath C., Godin B, & Khan A. (2022). CD44 receptor targeted nanoparticles augment immunity against tuberculosis in mice. Journal of controlled release : official journal of the Controlled Release Society, 349, 796-811.

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Western Blot Analysis of THOP Protein

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For the western blot, 1 μg of recombinant TOP and 30 μg of cell extract proteins per lane were separated by 12% SDS/PAGE and electrotransferred to a PVDF membrane Hybond‐P (GE Healthcare) and then blotted with primary antibodies according to the manufacturer's instructions anti‐24.15 (anti‐THOP, Proteimax Biotechnology, São Paulo, Brazil) or anti‐GAPDH (Proteimax Biotechnology) detected by an anti‐rabbit secondary antibody (T2767, Invitrogen Corporation, Carlsbad, CA, USA). Data were analyzed by western blot densitometry using imagej software 61.

Icimoto M.Y., Ferreira J.C., Yokomizo C.H., Bim L.V., Marem A., Gilio J.M., Oliveira V, & Nantes I.L. (2017). Redox modulation of thimet oligopeptidase activity by hydrogen peroxide. FEBS Open Bio, 7(7), 1037-1050.

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Immunofluorescence Imaging of LRRK2 and Mitochondria

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Fixed BV2 cells were permeablized by 0.1% Triton X-100 in Dulbecco’s phosphate buffered saline (DPBS) for 5 min at room temperature (RT). Coverslips were incubated with a blocking buffer composed of 3% bovine serum albumin and 1% goat serum in DPBS for 1 h. After the blocking step, the mouse monoclonal anti-LRRK2 antibody and rabbit polyclonal anti-Tom20 antibody in blocking buffer were added to cells for 8 h at 4°C followed by the incubation with Alexa Fluoro 488 conjugated anti-mouse (A-11001, Invitrogen) and Texas Red labeled anti-rabbit (T-2767, Invitrogen) secondary antibodies in blocking solution at RT for 2 h. BV2 cells on the inverted coverslip were mounted using ProLong Gold (P36930, Invitrogen) and five images were captured using a Zeiss LS55 confocal microscope operating in the Airyscan mode. BV2 cells expressing GS (n = 10) or transfected cells not expressing GS (n = 4) were selected and their mitochondria (n = 2–20/cell, total 34–107) were analyzed using the Mito-Morphology Macro of Image J software as previously described (Ho et al. 2018 (link)).

Ho D.H., Lee H., Son I, & Seol W. (2019). G2019s LRRK2 promotes mitochondrial fission and increases TNFα-mediated neuroinflammation responses. Animal Cells and Systems, 23(2), 106-111.

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