Alexa fluor 594 conjugated anti mouse

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Alexa Fluor 594-conjugated anti-mouse is a fluorophore-labeled secondary antibody used to detect and visualize mouse primary antibodies in various immunological techniques. It is a conjugate of the Alexa Fluor 594 dye and an anti-mouse antibody.

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18 protocols using alexa fluor 594 conjugated anti mouse

Quantifying Cx43 Immunoreactivity in Cardiomyocytes

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Sections were first fixed in 4% paraformaldehyde for 15 min. After that they were treated with 0.1% Triton X-100 for 1h and afterwards blocked by goat serum for 1h at room temperature. Specimens were incubated with primary antibodie of Cx43 (Santa Cruz, sc13558, lot# k2408, 1: 100) diluted with PBS for 3h at 37°C. After washing 3 times with PBS, specimens were incubated with secondary antibody 1:500 Alexa Fluor 594 conjugated anti-mouse (Invitrogen) for 1h at room temperature. Finally all sections were covered with cover slip and observed under confocal microscope. Cx43 immunoreactivity at the gap junctions in each image was quantified and normalized to the area of the respective cardiomyocyte using Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, MA, USA). The relative Cx43 immunoreactivity levels of 18M, OVX or OVX+E₂ rats that were presented in the column were normalized to 6M or Sham group.

Wang N., Sun L.Y., Zhang S.C., Wei R., Xie F., Liu J., Yan Y., Duan M.J., Sun L.L., Sun Y.H., Niu H.F., Zhang R, & Ai J. (2015). MicroRNA-23a Participates in Estrogen Deficiency Induced Gap Junction Remodeling of Rats by Targeting GJA1. International Journal of Biological Sciences, 11(4), 390-403.

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Immunostaining of E-cadherin, Vimentin, and β-Catenin

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Cells were seeded on 15‐mm glass coverslips (Paul Marienfeld GmbH & Co. KG, Lauda‐Königshofen, Germany) to proper confluent, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.05% Triton X for 5 min. The fixed cells were incubated with blocking buffer (3% BSA in PBS) at room temperature for 1 h, with primary antibodies (diluted in 1% BSA in PBS) overnight at 37 °C, and then incubated with secondary antibodies for 1 h at room temperature in the dark. The primary antibodies used include anti‐E‐cadherin (#610182) from BD Transduction Laboratories; antivimentin (#M7020) from Dako; and anti‐β‐Catenin (#8480) from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies are Alexa Fluor 488‐conjugated anti‐mouse (#A11029) and Alexa Fluor 594‐conjugated anti‐mouse (#A11032) from Invitrogen (Carlsbad, CA, USA). For F‐actin staining, rhodamine phalloidin (#R415; Life Technologies) was used directly after blocking. The stained coverslips were mounted onto glass slides using Vectashield mounting medium with DAPI (#H‐1200) from Vector Laboratories (Burlingame, CA, USA). Images were taken using Nikon (Minato, Tokyo, Japan) A1R confocal microscope. To quantify immunofluorescence (IF) expression of E‐cadherin and vimentin, mean fluorescence intensity was measured by using the automated measurement function in the nikon software.

Tan M., Asad M., Heong V., Wong M.K., Tan T.Z., Ye J., Kuay K.T., Thiery J.P., Scott C, & Huang R.Y. (2019). The FZD7‐TWIST1 axis is responsible for anoikis resistance and tumorigenesis in ovarian carcinoma. Molecular Oncology, 13(4), 757-780.

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Visualizing DNA Replication Foci

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DNA replication foci were visualized by incorporation of chlorodeoxyuridine (CldU) and iododeoxyuridine (IdU). Briefly, cells were labeled for 1 h with 10 μM CIdU and then 10 μM IdU (Chemos GmbH). Primary anti-CldU (Abcam) and then Alexafluor 488-conjugated anti-rat (Invitrogen) antibodies were added to the slides, and incubated for 1 h respectively. Then, primary mouse anti-IdU (Sigma) and then Alexafluor 594-conjugated anti-mouse (Invitrogen) antibodies were added to the slides, and incubated for 1 h respectively. Images were visualized with a laser confocal microscope.

Gou W.F., Shen D.F., Yang X.F., Zhao S., Liu Y.P., Sun H.Z., Su R.J., Luo J.S, & Zheng H.C. (2015). ING5 suppresses proliferation, apoptosis, migration and invasion, and induces autophagy and differentiation of gastric cancer cells: a good marker for carcinogenesis and subsequent progression. Oncotarget, 6(23), 19552-19579.

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Immunofluorescence Imaging of HA-CD147 and Flag-MCT1

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Example 9

Immunofluorescence was performed as described [10]. Briefly, HeLa cells stably expressing sh_CRBN or sh_scramble Were grown on glass coverslips and transfected with constructs expressing HA-CD147 and/or Flag-MCT1 using Lipofectamine (Invitrogen). Primary antibodies were anti-HA (mouse) and anti-FLAG (rabbit). Alexa Fluor 488 conjugated anti-rabbit and Alexa Fluor 594 conjugated anti-mouse (Invitrogen) were used as secondary antibodies. DAPI was used to counterstain DNA. Where indicated, ER tracker blue white DPX (Life technologies) was added to cells in culture for 30 min before fixation. Images were taken using the laser-scanning confocal microscope FluoView FV10i (Olympus). For quantification, 100 cells for each condition were analyzed in three independent experiments.

US10987345B2. Method for assessing the efficacy of IMiDs and composition or combination for use in treating IMiD sensitive diseases (2021-04-27). Klinikum Rechts Der Isar Der Technischen Universitat Munchen [DE]. Inventors: Florian Bassermann [DE], Ruth Eichner [DE], Vanesa Fernandez-Saiz [DE].

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Immunocytochemistry for Stem Cell Lineages

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Immunocytochemistry was used to detect osteocalcin and aggrecan localization in control and scaffolds after 21 days of osteogenic and chondrogenic differentiations, respectively. Likewise, cells were also immunostained by neural-specific markers β-III tubulin and MAP2 after 6 days of neuronal induction. Cells were fixed, permeabilized, and blocked as mentioned earlier and incubated with Alexa Fluor 488-conjugated rabbit polyclonal anti-osteocalcin (1:100; Biospes, China) overnight at 4°C. Similarly, overnight incubations at 4°C with the antibodies rabbit polyclonal anti-aggrecan (1:100; Biospes), mouse monoclonal anti-β-III tubulin (1:100; Santa Cruz, USA), and mouse monoclonal anti-MAP2 (1:100; Santa Cruz) were followed by 4 hour incubation with iFluor^® 488-conjugated anti-rabbit (1:200; Biospes), FITC-conjugated anti-mouse (1:200; Santa Cruz), and Alexa Fluor 594-conjugated anti-mouse (1:200; Invitrogen) secondary antibodies, respectively, at room temperature in darkness. After PBS washing, the coverslips were mounted on glass slides with DAPI ProLong Gold antifade solution (Invitrogen). All images were obtained using the same photographic parameters of exposure and speed in an inverted fluorescence microscope (Carl Zeiss) with Axio Vision 4.0 image analysis system.

Das K., Madhusoodan A., Mili B., Kumar A., Saxena A., Kumar K., Sarkar M., Singh P., Srivastava S, & Bag S. (2017). Functionalized carbon nanotubes as suitable scaffold materials for proliferation and differentiation of canine mesenchymal stem cells. International Journal of Nanomedicine, 12, 3235-3252.

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Plasmid Cloning and Antibody Characterization

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Details of the plasmids cloned for the study are available on request.
Plasmids that have been previously described include: NLS-MKL1 (ref. 5 (link)); MKL1(2-263)-2GFP-N3 (RPEL), MC-pEGFP-C1 and pET41a-3CΔ-MKL1(2-261)11 (link); MKL1(fl)-C471 (ref. 7 (link)); and SRF-VP16 (ref. 36 (link)). RanBPI-ΔNES-pEGFP-C1 and Smad4-pEGFP-C1 were kind gifts from Caroline Hill, pQE-RanQ69L from Maarten Fornerod and pRL-TK was from Promega.
The following antibodies were from Sigma: Flag (M2, F1804, 1:300), haemagglutinin (HA-7, G21234, 1:300), Ipoβ (31H4, I2534, 1:2,000) and Tubulin (B512, T6074, 1:2,000). MKL1 (C-19, SC-21558, 1:100) antibody was from Santa Cruz Biotechnology and Ddx19 (NB100-760, 1:2,000) was from Novus Biologicals. The following secondary antibodies were from Invitrogen: horseradish peroxidase (HRP)-conjugated anti-mouse (G21040, 1:5,000), HRP-conjugated anti-rabbit (G21234, 1:5,000), HRP-conjugated anti-goat (A9452, 1:5,000), Alexa-Fluor-488-conjugated anti-mouse (A21155, 1:500), Alexa-Fluor-488-conjugated anti-rabbit (A11008, 1:500), Alexa-Fluor-594-conjugated anti-mouse (A11005, 1:500), Alexa-Fluor-594-conjugated anti-rabbit (A11012, 1:500) and Alexa-Fluor-594-conjugated anti-goat (A11080, 1:500).

Rajakylä E.K., Viita T., Kyheröinen S., Huet G., Treisman R, & Vartiainen M.K. (2015). RNA export factor Ddx19 is required for nuclear import of the SRF coactivator MKL1. Nature Communications, 6, 5978.

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Immunohistochemical Analysis of Tissue Samples

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Immunohistochemical analyses were conducted, as previously reported^{32 (link)}. In brief, tissue sections were incubated with primary antisera (diluted 1:500) and then with one of the following antibodies: Alexa Fluor 488-conjugated anti-mouse (RRID: AB_2534069); anti-rabbit IgG (RRID: AB_2576217); Alexa Fluor 594-conjugated anti-mouse (RRID: AB_2534073); and anti-rabbit IgG (RRID: AB_2534079) (diluted 1:500; Invitrogen) for immunofluorescent detection. We examined the stained sections under a fluorescence microscope (Leica DM6000 B, Leica Microsystems) and a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems).

Kawano-Yamashita E., Koyanagi M., Wada S., Saito T., Sugihara T., Tamotsu S, & Terakita A. (2020). The non-visual opsins expressed in deep brain neurons projecting to the retina in lampreys. Scientific Reports, 10, 9669.

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Imaging Cell Proliferation and Apoptosis in Zebrafish Embryos

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Briefly, Tg(cmyb:EGFP) embryos were incubated with 10 mM BrdU (Sigma) for 30 minutes and incubated with egg water for another 2 hours, then fixed in 4% paraformaldehyde (PFA) at 3 dpf. After dehydration and rehydration, the embryos were treated with Proteinase K (10 mg/ml, Finnzyme) for 30 minutes and re-fixed in 4% PFA for 30 minutes. After blocking with blocking buffer (2 mg/ml BSA, 10% FBS, 0.3% Triton-X100 and 1% DMSO in PBST), the embryos were stained with rabbit anti-GFP (Invitrogen) primary antibody and Alexa Fluor 488-conjugated anti-rabbit (Invitrogen) secondary antibodies. The embryos were fixed again, treated with Proteinase K for the second time, and re-fixed in 4% PFA. The embryos were then incubated with 2 N HCl for 1 hour and stained with mouse anti-BrdU (Roche) and rabbit anti-GFP (Invitrogen) antibodies. Finally, Alexa Fluor 594-conjugated anti-mouse and Alexa Fluor 488-conjugated anti-rabbit (Invitrogen) secondary antibodies were used. Images were taken using Olympus FV 1000 confocal microscopy equipped with the FV10-ASW version3 software.
Terminal transferase UTP nick end labeling (TUNEL) was performed using the In Situ Cell Death Detection Kit, TMR red (Roche) according to the manufacturer's recommendations.

Yuan H., Zhang T., Liu X., Deng M., Zhang W., Wen Z., Chen S., Chen Z., de The H., Zhou J, & Zhu J. (2015). Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish. Scientific Reports, 5, 9011.

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Immunofluorescence Visualization of HA-CD147 and Flag-MCT1

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Example 9

Immunofluorescence was performed as described [10]. Briefly, HeLa cells stably expressing sh_CRBN or sh_scramble were grown on glass coverslips and transfected with constructs expressing HA-CD147 and/or Flag-MCT1 using Lipofectamine (Invitrogen). Primary antibodies were anti-HA (mouse) and anti-FLAG (rabbit). Alexa Fluor 488 conjugated anti-rabbit and Alexa Fluor 594 conjugated anti-mouse (Invitrogen) were used as secondary antibodies. DAPI was used to counterstain DNA. Where indicated, ER tracker blue white DPX (Life technologies) was added to cells in culture for 30 min before fixation. Images were taken using the laser-scanning confocal microscope FluoView FV10i (Olympus). For quantification, 100 cells for each condition were analyzed in three independent experiments.

US10398688B2. Method for assessing the efficacy of IMiDs and composition or combination for use in treating IMiD sensitive diseases (2019-09-03). Klinikum Rechts Der Isar Der TU München [DE]. Inventors: Florian Bassermann [DE], Ruth Eichner [DE], Vanesa Fernandez-Saiz [DE].

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Immunofluorescence Staining of ACSL4 and Calnexin

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HT-1080 cells were seeded on 8-well chambered slides (Fisher Scientific) at 0.05 million cells per well and let grow overnight. Cells were washed with PBS twice and fixed with 4% paraformaldehyde for 30 min at room temperature at dark. Cells were than permeabilized by three washes with PBST (PBS with 0.1% Triton X-100). Cells were blocked with 5% normal goat serum in PBST for 1 hr at room temperature. Cells were stained with mouse anti-ACSL4 antibody (Invitrogen) 1:250 and rabbit anti-Calnexin antibody (Invitrogen) 1:333 overnight at 4 °C. Cells were washed three times with PBST and stained with Alexafluor 594-conjugated anti-mouse (Invitrogen) 1:500 and Alexafluor 488-conjugated anti-rabbit antibody (Invitrogen) 1:500 at room temperature for 1 hr. Cells were washed three times with PBST. Slides were slightly dried and added with one drop of antifade mountant with DAPI (Invitrogen) and covered with cover glass. Slides were dried overnight and imaged using Zeiss LSM 800 confocal microscope.

Ji X., von Rosenvinge E.C., Johnson W.W., Armstrong R.N, & Gilliland G.L. (1996). Location of a potential transport binding site in a sigma class glutathione transferase by x-ray crystallography.. Proceedings of the National Academy of Sciences of the United States of America, 93(16), 8208-8213.

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