F2761

Manufactured by Thermo Fisher Scientific

Sourced in United States

The F2761 is a laboratory instrument designed for temperature and humidity monitoring. It is capable of measuring and recording environmental conditions within a controlled laboratory setting.

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10 protocols using f2761

Whole-mount Immunofluorescence of β-catenin and Nanog in Embryos

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Whole-mount immunofluorescence of β-catenin was carried out based on the standard protocol using mouse anti–β-catenin antibody (C7267, Sigma-Aldrich, Saint Louis, Missouri, 1:500 dilution). Different stages of embryos were fixed overnight with 4% PFA in PBS at 4 °C, washed with PBST (0.1% Triton X-100 added), and permeabilized with distilled water for 1 hour. The FITC conjugated goat anti-mouse antibody (F2761, Thermo, Waltham, Massachusetts, 1:500) was used as secondary antibody. After 4 times of washing in PBST with 0.1% Triton X-100, embryos were incubated in DAPI solution (5 μg/mL in PBST) for 1 hour at room temperature. Then, embryos were washed and mounted at animal view for observation. Immunostained embryos were scanned at Z-stack using a Leica SP8 confocal microscope as described previously [95 (link)].
For Nanog immunostaining, rabbit anti-Nanog polyclonal antibody was customized by ABclone (Wuhan, China) using a recombinant Nanog protein (1:200 dilution). Embryos were fixed overnight with 4% PFA in PBS at 4 °C and transferred into 30% sucrose/PBS, incubated at 4 °C for 1 day, mounted in mounting medium (4583, Tissue-Tek OCT Compound, Sakura, Torrance, California), and then cryosectioned. Cryosections of 10-μm thickness were used for immunostaining. Signals were photographed using a Leica SP8 confocal microscope as described previously [96 (link)].

He M., Zhang R., Jiao S., Zhang F., Ye D., Wang H, & Sun Y. (2020). Nanog safeguards early embryogenesis against global activation of maternal β-catenin activity by interfering with TCF factors. PLoS Biology, 18(7), e3000561.

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Immunofluorescence Analysis of SARS-CoV-2 Proteins

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Ten-micrometer-thick cryosections were double-stained using a monoclonal mouse anti-SARS-CoV-2 antibody (1:1000 dilution, #MBS569903; MyBioSource, San Diego, CA, USA) and one of the following antibodies: polyclonal rabbit anti-pan cytokeratin antibody (1:100 dilution, #ab9377; Abcam), monoclonal rabbit anti-CD14 antibody (1:100 dilution, #ab18332; Abcam), polyclonal rabbit anti-ACE2 (1:100 dilution, recognizing both short and long forms of ACE2; #PK-AB718-3217, PromoCell, Heidelberg, Germany), monoclonal rabbit anti-ICAM-1 (1:100 dilution, #ab109361, Abcam). Mouse monoclonal and rabbit monoclonal or polyclonal isotype antibodies (#ab18469, #ab172730 and #ab15348; Abcam) functioned as negative controls. In the secondary step, FITC-conjugated polyclonal goat anti-mouse antibodies (1:200 dilution #F2761, ThermoFisher Scientific) were combined with Texas Red-conjugated polyclonal donkey anti-rabbit antibodies (1:100 dilution, #ab6800, Abcam). In the tertiary step, FITC-conjugated polyclonal donkey anti-goat antibodies were added (1:200 dilution, #A16006, ThermoFisher Scientific). DAPI (#D9542, Sigma-Aldrich) was used to counterstain cell nuclei. Slides were mounted with Fluoroshield™ (#F6182, Sigma-Aldrich) and analyzed using a Leica (TCS SPE) confocal microscope and Leica Las X v3.7.2.22383 software.

Van Cleemput J., van Snippenberg W., Lambrechts L., Dendooven A., D’Onofrio V., Couck L., Trypsteen W., Vanrusselt J., Theuns S., Vereecke N., van den Bosch T.P., Lammens M., Driessen A., Achten R., Bracke K.R., Van den Broeck W., Von der Thüsen J., Nauwynck H., Van Dorpe J., Gerlo S., Maes P., Cox J, & Vandekerckhove L. (2021). Organ-specific genome diversity of replication-competent SARS-CoV-2. Nature Communications, 12, 6612.

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Immunofluorescent Assay of Runx2 and Collagen I

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An immunofluorescent assay was performed for Runt-related transcription factor 2 (Runx2) (ab76956; Abcam, Cambridge, UK) and collagen I (ab6308; Abcam) on days 1, 3, 5 and 7. The cells were fixed, permeabilized, blocked, and incubated with primary antibodies. Mouse monoclonal Runx2 antibody was diluted at 1:50 and was incubated overnight incubation at 4°C. Mouse monoclonal collagen I antibody was diluted at 1:67 and was incubated overnight incubation at 4°C. The cultures were incubated with fluorescein isothiocyanate-conjugated secondary antibody (F2761; Thermo Fisher Scientific, Inc., Waltham, MA, USA) diluted at 1:100 and incubated for 2 h at room temperature. The washed cells were stained with 4′,6-diamidino-2-phenylindole (DAPI). Analyses were performed using a fluorescence microscope (Axiovert 200; Zeiss GmbH, Jena, Germany).

Lee H, & Park J.B. (2017). Evaluation of the effects of dimethylsulphoxide on morphology, cellular viability, mRNA, and protein expression of stem cells culture in growth media. Biomedical Reports, 7(4), 291-296.

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Quantifying E-cadherin and α-SMA in HK-2 Cells

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E-cadherin and α-SMA expression was assessed via immunofluorescence in HK-2 cells by plating these cells in 96-well plates, washing them with PBS twice, and then fixing them using 4% paraformaldehyde. Subsequently, 5% BSA in PBS was used to block the cells for 30 min at room temperature, after which they were incubated with primary anti-α-SMA (1:200, ab7817; Abcam) and anti-E-cadherin (1:50, ab40772; Abcam) antibodies at room temperature for 1 h. Cy3-conjugated goat anti-rabbit secondary antibody (1:200, A10520; Thermo Fisher Scientific, Inc.) and FITC-conjugated goat anti-mouse secondary antibody (1:200, F2761; Thermo Fisher Scientific, Inc.) were then incubated with these cells for 1 h at 37°C, after which Hoechst 33258 (Thermo Fisher Scientific, Inc.) was used for nuclear staining at room temperature for 5 min, and a Thermo Fisher Scientific ArrayScan HCS reader was used for image acquisition, with the Thermo HCS Studio™ 2.0 Cell Analysis Program was used for subsequent data analysis.

Wang Q., Tao Y., Xie H., Liu C, & Liu P. (2021). MicroRNA-101 inhibits renal tubular epithelial-to-mesenchymal transition by targeting TGF-β1 type I receptor. International Journal of Molecular Medicine, 47(6), 119.

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Immunohistochemical Analysis of PDZK1 in HCC

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Slices of HCC and normal tissue were dewaxed, dehydrated, and rehydrated. Primary antibody against PDZK1(10507-2-AP, PROTEINTECH, USA) was added to the sections and incubated overnight at 4 °C. Then, biotinylated secondary antibody (F2761; Thermo Fisher Scientific, Waltham, MA, USA) was applied according to the protocol of the SP-immunohistochemistry (IHC) test [38 (link)].

Gao H., Gao Z., Liu X., Sun X., Hu Z., Song Z., Zhang C., Fei J, & Wang X. (2024). miR-101-3p-mediated role of PDZK1 in hepatocellular carcinoma progression and the underlying PI3K/Akt signaling mechanism. Cell Division, 19, 9.

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Immunohistochemical Analysis of Rac1 Expression

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The paraffin-embedded sections were deparaffinized and rehydrated. Then, antigens were retrieved in 10 mM boiled citrate buffer (pH 6.0, Beijing Solarbio Science & Technology Co., Ltd., China) for 30 min. Following permeabilization with 0.3% Triton X-100 in PBS for 30 min, the sections were incubated with PBS containing 5% goat serum and 0.3% Triton X-100 for 2 h to block non-specific binding. To evaluate the expression of Rac1, the sections were incubated with mouse anti-RAC1 (1:200, 05-389, Sigma–Aldrich) overnight at 4°C, followed by incubation with FITC-conjugated goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (1:500, F-2761, Invitrogen, Thermo Fisher Scientific, Inc.) for 2 h at room temperature. After being washed with PBS, the nuclei were counterstained with 4′-DAPI (Sigma–Aldrich) for 5 min. The primary antibody was diluted with blocking buffer, and isotype control antibodies were used as a negative control (Mouse IgG Isotype Control, 10400C, Invitrogen, Thermo Fisher Scientific, Inc.). Images were captured with a Pannoramic MIDI digital microscope (3DHISTECH, Budapest, Hungary) and were analysed using CaseViewer (3DHISTECH, Budapest, Hungary).

Diao M., Zhou J., Tao Y., Hu Z, & Lin X. (2022). RAC1 is involved in uterine myometrium contraction in the inflammation-associated preterm birth. Reproduction (Cambridge, England), 164(4), 169-181.

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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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Immunofluorescence Staining of FLAG-Tagged Proteins

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Cells were resuspended in 50 μl of FBS (Gibco) and spread on coated slides to air dry. Cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 20 min at room temperature, washed with DPBS, permeabilized with 0.5% Triton X-100/PBS for 3 min at room temperature, and blocked in 5 mg/ml BSA (Sigma-Aldrich) for 30 min before being probed with a FLAG.Tag (1:200, F1804, Sigma-Aldrich) or p65 (1:200, 10745-1-AP, Proteintech) primary antibody followed by an anti-mouse-fluorescein secondary antibody (1:200, F-2761, Invitrogen) or an anti-rabbit Alexa Fluor-488 (1:200, 21206, Invitrogen). Fluorescence images were visualized with a Zeiss LSM880 laser scanning confocal system (Carl Zeiss). Images were processed using ZEN 2009 Light Edition software (Carl Zeiss).

Hwang W.L., Lan H.Y., Cheng W.C., Huang S.C, & Yang M.H. (2019). Tumor stem-like cell-derived exosomal RNAs prime neutrophils for facilitating tumorigenesis of colon cancer. Journal of Hematology & Oncology, 12, 10.

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Immunofluorescence Analysis of HOX Proteins

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Cells were plated at a density of 1 × 10⁵ cells/well in 12‐well plates with coverslips overnight and then treated with 60 μmol/L HXR9 or CXR9 for 2 hours. After being fixed in 3.7% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X‐100 for 5 minutes, the coverslips were blocked in 5% normal serum and then incubated in primary antibody dilutions overnight at 4°C. The primary antibodies used were anti‐PBX (sc‐28313 at 1:50, mouse; Santa Cruz Biotechnology), anti‐HOXB7 (#40‐2000 at 1:50, rabbit; Thermo Fisher Scientific, Waltham, MA, USA), anti‐HOXC6 (#PA5‐65913 at 4 μg/mL, rabbit; Invitrogen, Carlsbad, CA, USA), and anti‐HOXC8 (ab86236 at 1:50, rabbit; Abcam). Then, coverslips were incubated in the appropriate fluorophore‐conjugated secondary antibody dilutions and counterstained with DAPI (D523 at 300 nmol/L; Invitrogen) dilution. The secondary antibodies used were FITC‐conjugated goat antimouse IgG (F2761 at 1:50; Invitrogen) and TRITC‐conjugated goat antirabbit IgG (T2769 at 1:50; Invitrogen). Immunofluorescence was visualized by Zeiss scanning microscope (Zeiss Germany, Oberkochen, Germany).

Shen L., Zhou T., Du Y., Shi Q, & Chen K. (2019). Targeting HOX/PBX dimer formation as a potential therapeutic option in esophageal squamous cell carcinoma. Cancer Science, 110(5), 1735-1745.

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Immunocytochemistry of Stem Cell Markers

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Five thousand cells per well were seeded on to coverslips, washed in 100% ethanol and placed in 24-well plates. The cells were incubated until they achieved 50–60% confluency. The medium was removed, and the cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 min. PFA was removed and the cells were washed with wash buffer, tris buffered saline (TBS), and blocked with 1% BSA to prevent non-specific protein binding. The blocking buffer was removed and the cells were washed with TBS. Primary antibodies survivin (sc-17779), SOX2 (Invitrogen-MA1-014) and Nestin (Invitrogen-MA1-110) were added to the cells and were incubated overnight at 4 °C. Staining was visualized with mouse IgGκ conjugated to fluorescein isothiocyanate (FITC), Goat Anti-Mouse IgG(H + L) cross-adsorbed secondary antibody ((Invitrogen -F2761) and (Invitrogen- P-852) and counterstained with DAPI. The specimens were mounted on microscope slides and visualized. Fluorescence observation and photo-documentation were obtained using an inverted fluorescent microscope [62 (link)].

Warrier N.M., Krishnan R.K., Prabhu V., Hariharapura R.C., Agarwal P, & Kumar P. (2022). Survivin Inhibition by Piperine Sensitizes Glioblastoma Cancer Stem Cells and Leads to Better Drug Response. International Journal of Molecular Sciences, 23(14), 7604.

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