Samples were incubated overnight at 4°C with the following primary antibodies: mouse anti‐m3G (1:50; Santa Cruz Biotechnology, https://www.scbt.com ), mouse anti‐U2B″ (1:20; LS Bio, https://www.lsbio.com ) and rat anti‐RNA pol II (elongated form, hyperphosphorylated Ser2 of the C‐terminal domain; 1:100; Chromotek, https://www.chromotek.com ) in 0.01% acBSA in PBS, pH 7.2. After rinsing with PBS, the samples were incubated for 1 h at 37°C with the following secondary antibodies: anti‐mouse Alexa 546 (ThermoFisher Scientific), anti‐rat Alexa 488 (ThermoFisher Scientific) or anti‐rabbit Alexa 488 (ThermoFisher Scientific) 1:500 in 0.01% acBSA in PBS, pH 7.2. Next, the slides were rinsed with PBS, pre‐incubated with 2% BSA in PBS, pH 7.2, for 15 min, and incubated overnight at 4°C with the anti‐Sm ANA No. 5 antibody diluted 1:300 in 0.2% acBSA in PBS, pH 7.2. After rinsing with PBS, the samples were incubated for 1 h at 37°C with the appropriate respective anti‐human antibody: Alexa 488 at 1:750 or TRITC, at 1:200 in 0.01% acBSA in PBS, pH 7.2.
Anti mouse alexa546
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-mouse-Alexa546 is a fluorescently labeled secondary antibody that binds to mouse primary antibodies. The Alexa Fluor 546 dye attached to the secondary antibody emits red fluorescence when excited, enabling visualization and detection of target proteins or molecules in various biological applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488 anti rabbit, by Thermo Fisher Scientific (6 mentions)
Anti mouse alexa fluor 488, by Thermo Fisher Scientific (4 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Anti mouse cy5, by Jackson ImmunoResearch (2 mentions)
Bu1 75, by Bio-Rad (2 mentions)
Anti rat dylight549, by Jackson ImmunoResearch (2 mentions)
Anti rabbit dylight549, by Jackson ImmunoResearch (2 mentions)
Anti rat alexa633, by Thermo Fisher Scientific (2 mentions)
Anti chicken 488, by Jackson ImmunoResearch (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Rat anti rna pol 2, by Proteintech (1 mentions)
Anti rat alexa 488, by Thermo Fisher Scientific (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
Leica dmi6000, by Leica Microsystems (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Thermo Fisher Scientific (1 mentions)
Ctnt antibody, by Thermo Fisher Scientific (1 mentions)
Bz x800e, by Keyence (1 mentions)
9 protocols using anti mouse alexa546
1
Immunofluorescence Staining of Cellular Structures
2
Chromosome Segregation Under Cadmium Stress
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The segregation of chromosomes under Cd-mediated stress conditions was analyzed with the use of immunostaining followed by fluorescence microscopy, as was previously described by Rabitsch et al. [61 (link)]. Shortly, a yeast strain with GFP-labeled chromosome II (JG 15,457 cen2(D107):KanR-ura4+-lacO his7+::lacI-GFP) in which a chromosome locus close to the centromere is visualized through a specific binding of the LacI-GFP fusion repressor to the lacO tandem repeats sequence was grown in YES medium to OD600 = 1. Cells were incubated with 0, 50, and 100 μM of Cd for 3 h; fixed with 4% paraformaldehyde (PFA) in PEMS (PEM + 1.2 M sorbitol); and stained with primary TAT1 mouse monoclonal anti-tubulin (1:200) and rabbit polyclonal anti-GFP (Thermo Fisher Scientific; Waltham, USA, 1:400) antibodies. After 16–24 h on a wheel at RT and 3 washing steps with PEMBAL, the samples were resuspended in PEMBAL (PEM + 1% BSA + 0.1% NaN3) containing anti-mouse-Alexa546 and anti-rabbit-Alexa488 (Thermo Fisher Scientific; Waltham, USA) both diluted 1:500 and incubated as before. After washing, the samples were mounted on poly-L-lysine-coated cover slips in Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA) to visualize the DNA. Analyses were performed on an inverted fluorescent microscope equipped with a digital camera (Leica DMI 6000, Leica microsystems, Wetzlar, Germany).
3
Immunofluorescence Analysis of ACE2 and SARS-CoV-2 Spike Protein
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For IFA, CMTs were stained with cTnT antibody (Thermo Fisher) (1:250), CD31 (monoclonal mouse IgG1, clone 9G11) (R&D) (1:250), ACE2 (polyclonal antibody) (Bioss, Woburn, MA, USA) (1:250), S protein (ab272504), Abcam (1:250) or monoclonal mouse antibody provided by Dr. Hisashi Arase (Osaka University) (1:50), with DAPI (4‘,6-diamidino-2-phenylindole) (Thermo Fisher) (1:1000). Anti-mouse Alexa 546 (Thermo Fisher), anti-rabbit Alexa 488 (Thermo Fisher) and anti-mouse Alexa Fluor 488 (Thermo Fisher) were used as secondary antibodies. The tissues were photographed with an all-in-one fluorescence microscopic system, BZ-X800E (Keyence, Osaka, Japan) Combined Z-stack and sectioning functions. All results were confirmed with >2 repetitive independent experiments.
4
Detailed Immunohistochemistry of Heart Tissue Slices
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Thin paraffin sections of tissue slices were stained with hematoxylin–eosin using standard techniques. Immunohistochemistry was done on whole-mount heart slices that had been fixed in 4% paraformaldehyde for at least 24 h. The tissues were equilibrated with a graded series of 4, 15 and 30% sucrose in PBS, and were permeabilized with 1% Triton X-100 overnight. After blocking (3% BSA in PBS, 12 h), samples were incubated sequentially with primary antibodies (anti-α-actinin, A7811, anti-connexin-43, C6219, anti-α-smooth muscle actin, A5228, all Sigma-Aldrich, anti-vimentin, AB92547, Abcam, anti-N-cadherin, #610921, BD-Biosciences, all 1:100), and secondary antibodies (anti-rabbit-Alexa488, A21441, anti-mouse-Alexa546, A11030, both ThermoFisher, 1:100, combined with DNA-stain 1 µM TO-PRO-3) for 1 day each. Washing steps between all incubations used citrate-buffer (150 mM NaCl, 15 mM Na3-citrate, pH 7.2) supplemented with 1% BSA, 0.05% Triton X-100 and 3 mM NaN3. Slices were mounted in VectaMount AQ (Vector Laboratories). Confocal microscopy was performed at the bioimaging core facility of the Biomedical Center using an inverted Leica SP8X WLL microscope.
5
Immunostaining of Cells on Biomaterial Scaffolds
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Cells cultured on TCP, GelMA, and GelMA-PSS were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature, washed in PBS, blocked with 3% (w/v) bovine serum albumin in PBS, and permeabilized with 0.1% (v/v) Triton X-100 for 1 h at room temperature. Samples were incubated with primary antibodies (diluted in 1% BSA in PBS), including rabbit anti-Desmin (1:200, Abcam, Cambridge, UK) and mouse anti-MyHC (MF-20) (1:200, Developmental Studies Hybridoma Bank, Iowa City, IA, USA), overnight at 4 °C, washed 3 times in fresh PBS, and incubated with secondary antibodies, including anti-rabbit Alexa 488 (1:200, Thermo Fisher Scientific, Waltham, MA, USA) (diluted in 1% BSA in PBS), anti-mouse Alexa 546 (1:200, Thermo Fisher Scientific, Waltham, MA, USA), as well as with an anti-phalloidin (1:200, Thermo Fisher Scientific, Waltham, MA, USA) antibody for 1 h at room temperature. The nuclei were stained with Hoechst 33342 (2 mg/mL; Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at room temperature. All fluorescence images were acquired by using an inverted microscope (Eclipse Ti-U, Nikon, Tokyo, Japan) at the Soonchunhyang Biomedical Research Core-Facility of the Korea Basic Science Institute (KBSI).
6
Histological Analysis of 3D Cardiac Microtissues
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Three-dimensional cardiac microtissues were fixed in 4% PFA and embedded in paraffin. Sections (6-μm thickness) were prepared and stained with haematoxylin–eosin and Sirius red. For fluorescence microscopy, 3D cardiac microtissues were stained for cTnT (Thermo Fisher) (1:500), calponin (anti-calponin1 antibody, clone EP798Y) (Abcam, Cambridge, UK) (1:500) and CD31 (monoclonal mouse IgG1, clone 9G11) (R&D) (1:50) with DAPI (4′,6-diamidino-2-phenylindole) (Thermo Fisher). Anti-mouse Alexa 546 (Thermo Fisher), anti-rabbit Alexa 488 (Thermo Fisher) and anti-mouse Alexa Fluor 488 (Thermo Fisher) were used as secondary antibodies. The tissues were photographed with an all-in-one fluorescence microscopic system, Biorevo BZ-9000 (Keyence, Osaka, Japan).
7
Immunolabeling of Retinal Cell Types
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The following primary antibodies were used: anti-EGFP (chicken; Life Technologies, A10262; 1:500), rabbit anti-Rx2 (Reinhardt et al., 2015 (link); 1:500), anti-tagRFP (rabbit; Evrogen, AB233; 1:500), anti-Pax6 (rabbit; Hiss Diagnostics, PRB-278P; 1:200), PKCα (rabbit; Santa Cruz, sc-208; 1:200), anti-GS (mouse; Chemicon, MAB302; 1:500), anti-Sox2 (rabbit; Genetex, GTX101506; 1:500), anti-HuC/D (mouse; Thermo Fisher, A21271; 1:500), anti-recoverin (rabbit; Millipore, AB5585; 1:500), anti-Zpr-1 (mouse; Zebrafish International Resource Center; 1:500), anti-DsRed (rabbit; Clontech, 632496; 1:500), anti-BrdU (rat; AbD Serotec, BU1/75; 1:200). The following secondary antibodies were used: anti-mouse Cy5 (Jackson ImmunoResearch, 715-175-151), anti-chicken 488 (Jackson ImmunoResearch, 703-485-155), anti-rat DyLight549 (Jackson ImmunoResearch, 112-505-143), anti-rabbit DyLight549 (Jackson ImmunoResearch), anti-mouse Alexa546 (Life Technologies, A-11030) and anti-rat Alexa633 (Life Technologies, A21094). DAPI (Sigma-Aldrich, D9564) nuclear counterstaining was performed as described by Inoue and Wittbrodt (2011) (link).
8
Golgi Localization of Ion Channels
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To evaluate the localization of our proteins relative to Golgi apparatus, cells were treated with cell light Golgi-GFP (Life Technologies, Eugene, OR; 5 μl/10000 cells) 48 hours before fixation, simultaneously of cells starvation. Cells were fixed with paraformaldehyde 4% for 20 minutes, permeabilized with triton 0.5% and blocked for 30 minutes with BSA 3%. The cells are then incubated overnight with primary antibodies (Kv10.1 1/50, Santa Cruz Biotechnology, Inc., Heidelberg, Germany; Orai1 1/200, Sigma Aldrich, Saint-Quentin-Fallavier, France; SPCA2 1/100, Santa Cruz Biotechnology, Inc., Heidelberg, Germany), washed with PBS, incubated 1 hour with secondary antibodies conjugated with fluorophores (anti-mouse Alexa 546, 1/200, Life Technologies, Eugene, OR; anti-rabbit Alexa 549, 1/200, Thermo Fisher Scientific, Rockford, IL) and counterstained with DAPI to visualize the nuclei. Analyses were performed on a LSM 780 confocal microscope (Carl Zeiss) and analyzed with ZEN 2012 software. For immunofluorescence assay on human tissue, samples were stained with anti-Kv10.1 (1/50, Alomone Labs, Jerusalem, Israel) and TGN46 (1/100, Thermofisher Scientific).
9
Multimodal Labeling of Retinal Cells
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Fluorescence whole-mount in situ hybridization was essentially carried out as described previously (Souren et al., 2009 (link)). To determine the identity of atoh7-expressing cells in the INL, an anti-GS stain was performed in combination with the fluorescence atoh7 in situ as described by Inoue and Wittbrodt (2011 (link)). For immunohistochemistry, embryos were fixed overnight in 4% paraformaldehyde (PFA) in PTW at 4°C and mounted for cryosectioning. Antibody staining was performed as described by Inoue and Wittbrodt (2011 (link)), using the following primary antibodies (1:500): anti-GS (mouse; Chemicon, MAB302), anti-EGFP (chicken; Life Technologies, A10262), anti-DsRed (rabbit; Clontech, 632496), rabbit anti-Rx2 (Reinhardt et al., 2015 (link)), anti-PCNA (mouse; Santa Cruz, sc-56) and anti-BrdU (rat; AbD Serotec, BU1/75). The following secondary antibodies were used (1:500): anti-mouse Cy5 (Jackson ImmunoResearch, 715-175-151), anti-chicken 488 (Jackson ImmunoResearch, 703-485-155), anti-rat DyLight549 (Jackson ImmunoResearch, 112-505-143), anti-rabbit DyLight549 (Jackson ImmunoResearch), anti-mouse Alexa546 (Life Technologies, A-11030) and anti-rat Alexa633 (Life Technologies, A21094). DAPI (Sigma-Aldrich, D9564) nuclear counterstaining was performed as described by Inoue and Wittbrodt (2011 (link)).
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