Donkey anti mouse alexa fluor 555

Manufactured by Thermo Fisher Scientific

Sourced in United States

Donkey anti-mouse Alexa Fluor 555 is a secondary antibody conjugated with the Alexa Fluor 555 fluorescent dye. It is designed to detect and visualize mouse primary antibodies in various biological applications such as immunofluorescence, flow cytometry, and Western blotting.

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38 protocols using donkey anti mouse alexa fluor 555

Immunohistochemical Visualization of HuC/D

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Immunohistochemistry was performed essentially as previously described54 (link). To examine the HuC/D (Life Technologies, A21271), the embryos were first stained with HuC/D first antibody (20 μg/ml, 4°C, overnight) and were subsequently visualized by Alexa Fluor-555 donkey anti–mouse (Life Technologies, A-31570).

Shi Y., Zhang Y., Zhao F., Ruan H., Huang H., Luo L, & Li L. (2014). Acetylcholine serves as a derepressor in Loperamide-induced Opioid-Induced Bowel Dysfunction (OIBD) in zebrafish. Scientific Reports, 4, 5602.

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BrdU Labeling for Cell Proliferation Analysis

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For BrdU labeling, BrdU (Roche Diagnostics, Indianapolis, IN, United States; 1 nl, 30 mM) was injected into the pericardium of embryos. Consequently, the embryos were incubated for 1.5–2 h at 28.5°C. After three times of washing with PBST, the embryos were fixed using 4% PFA. After being treated with 2 N HCl for 1 h, the embryos were incubated with mouse anti-BrdU (Roche Diagnostics, United States; 1:50) and goat anti-GFP (Abcam, United States; 1:400) antibodies at 4°C overnight, and finally visualized by Alexa Fluor 555 donkey anti-mouse (Life Technology, Carlsbad, CA, United States; 1:400) and Alexa Fluor 488 donkey anti-goat (Life Technology, United States; 1:400) antibodies.

Zhang C., Huang R., Ma X., Chen J., Han X., Li L., Luo L., Ruan H, & Huang H. (2021). The Ribosome Biogenesis Factor Ltv1 Is Essential for Digestive Organ Development and Definitive Hematopoiesis in Zebrafish. Frontiers in Cell and Developmental Biology, 9, 704730.

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Immunohistochemical Analysis of Membrane dHACM

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For ease of maintaining tissue orientation during histological sectioning, membrane dHACM tissue samples (n = 3) were used for immunohistochemistry (IHC). Tissues were hydrated in 0.9% NaCl for 10 min, embedded in TissueTek optimum cutting temperature compound (Sakura Finetek), and cryosectioned at a thickness of 5 μm. Sections were fixed by incubation in chilled acetone for 10 min and dried at room temperature for at least 30 min. The sections were washed with phosphate-buffered saline (PBS), blocked, and incubated with primary antibodies (mouse anti-human collagen IV, 1:100; Abcam, mouse anti-human collagen I, 1:1,000; sheep anti-human HA, 1:100; Abcam) overnight at 4°C. After washing with PBS, sections were stained with a secondary antibody (Alexa Fluor^® 660 goat anti-mouse; Alexa Fluor 555 donkey anti-mouse; Alexa Fluor 647 donkey anti-sheep, respectively; 1:100; Life Technologies) for 30 min at room temperature and mounted in Fluorogel (Electron Microscopy Sciences).

Lei J., Priddy L.B., Lim J.J., Massee M, & Koob T.J. (2017). Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane. Advances in Wound Care, 6(2), 43-53.

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Immunohistochemical Analysis of TRPV5 and Calbindin

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Animals were injected with anesthesia cocktail (ketamine/xylazine/acepromazine, 50/5/0.5 mg/kg), and under deep anesthesia animals were perfusion fixed with 4% paraformaldehyde. After cryoprotection in 800 mosmol/L sucrose and freezing in Optimal Temperature Cutting (OCT) compound, 5‐μm sections were cut. Sections were incubated overnight at 4°C with anti‐anti‐TRPV5 (1:100, Alomone) or anti‐calbindin (1:500, Swant). Sections were incubated in secondary antibody at 1:2000 for 1 h at room temperature [Alexa Fluor 488 donkey anti‐rabbit (Life Technologies A21206) or Alexa Fluor 555 donkey anti‐mouse (Life Technologies A31570)]. All sections were mounted with ProLong Diamond Antifade Mountant (ThermoFisher Scientific P36970). Images were captured with a ZEISS AXIO Imager M2 fluorescent microscope.

Ferdaus M.Z., Gratreak B.D., Miller L., Si J., McCormick J.A., Yang C., Ellison D.H, & Terker A.S. (2019). WNK4 limits distal calcium losses following acute furosemide treatment. Physiological Reports, 7(17), e14195.

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Immunofluorescent Staining of Transfected Cells

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After transfection for 24 h, the cells were re-seeded on the coverslips in a 24-well cell culture plate of 50–60% confluency. The cells were fixed with 4% formalin for 10 min and blocked in PBS with 1% BSA and 0.5% Triton X-100 for 30 min at room temperature. Then, the cells were incubated with primary antibodies against CCL2 (Millipore, Burlington, MA, USA, cat#MABN712, 1:100), CCL3 (Acris, cat#PP038P2, 1:100), PU.1 (Abcam, Cambridge, UK, cat#ab88082, 1:100), or Flot1 (Abcam, cat#ab41927, 1:100) at 4 °C overnight. Secondary antibodies, namely Alexa Fluor 555 donkey anti-mouse (Life technologies, Carlsbad, CA, USA, Ref: A31570) and Alexa Fluor 555 donkey anti-rabbit (Life technologies, Ref: A31572), were applied at a 1:200 dilution for 1 hour at room temperature in the dark. The nuclei were stained with DRAQ5^TM at a 1:1000 dilution for 30 min at room temperature. After washing with PBS three times, the slides were mounted with a fluorescence-preserving VECTASHIELD^® HardSetTM Antifade Mounting Medium (Vector Laboratories, cat#H-1400). The images were taken with a Leica TCS SP8 confocal microscope.

Chen Y., Lin J., Schlotterer A., Kurowski L., Hoffmann S., Hammad S., Dooley S., Buchholz M., Hu J., Fleming I, & Hammes H.P. (2021). MicroRNA-124 Alleviates Retinal Vasoregression via Regulating Microglial Polarization. International Journal of Molecular Sciences, 22(20), 11068.

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Immunofluorescence Analysis of pATM and Ki-67

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HUVECs grown on glass coverslips were fixed with 4% paraformaldehyde, permeabilized in 0.25% Triton X-100 for 5 min, fixed with ice-cold methanol for 10 min, and blocked with a solution of 3% BSA and 0.05% Tween20, for 30 min at 37 °C. After washing, cells were incubated in PBS containing 3% BSA and 0.05% Tween20, for 2 h at 37 °C, with primary antibodies: mouse Phospho ATM (Serine 1981) Mab (1:200; Cell Signaling, Danvers, MA, USA #4526), and rabbit anti Ki-67 (1:200; ThermoFisher Scientific#RM-9106-S1 Waltham, MA, USA). Cells were washed and incubated in 0.05% Tween 20, 1% BSA, and Alexa Fluor 555 donkey anti-mouse, or Alexa Fluor 555 donkey anti-rabbit secondary antibodies (1:400; Life Technologies, Waltham, MA, USA) for 1 h at RT in humid chamber. Cells were washed and incubated with DAPI (1:2000; Sigma-Aldrich, St. Louis, MO, USA) before mounting the coverslips.
Images were acquired using a laser confocal microscope Olympus FV1200. pATM- and Ki-67-positive cells were counted by a blinded investigator. Mean values and standard deviation were generated from at least three biological replicates.

Merolle M., Mongiardi M.P., Piras M., Levi A, & Falchetti M.L. (2020). Glioblastoma Cells Do Not Affect Axitinib-Dependent Senescence of HUVECs in a Transwell Coculture Model. International Journal of Molecular Sciences, 21(4), 1490.

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Immunohistochemical Analysis of Embryonic Mouse Tissues

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For immunohistochemistry, heterozygous mice were fixed with 4% paraformaldehyde in PBS by intracardial perfusion. Tissues were post-fixed for 1h to overnight at 4°C. E14.5 mice were dated with E0.5 at time of plug detection. Embryos were drop fixed in 4% paraformaldehyde immediately after dissection. After post-fix, all tissue was washed extensively with PBS, cryopreserved in 30% sucrose in PBS overnight, embedded in OTC (Sakura Finetek, Torrance, CA), and frozen. Sections were cut at 20 μm on a cryostat and placed on slides. For immunostaining, sections were blocked in 10% goat serum and 0.25% triton-X in PBS for 1 h at RT. Sections were incubated with primary antibodies in block overnight at 4°C. Slides were washed 4 × 5 min in PBS containing 0.1% triton-X. Detection of antibodies was carried out using Alexa-fluor secondary antibodies (Life Technologies) diluted 1: 500 in blocking solution. Sections were washed, as above, and coverslipped. The following primary antibodies used were: rabbit anti-GFP (1:1,000, Life Technologies), goat anti-ChAT (1:500, Millipore), mouse anti-PAX6 (1:200, Developmental Studies Hybridoma Bank). Secondary antibodies: Alexa Fluor-488 donkey anti-rabbit (1:500, Life Technologies), Alexa Fluor-555 donkey anti-goat (1:500, Life Technologies), Alexa-Fluor-555 donkey anti-mouse (1:500, Life Technologies).

Cai X., Huang H., Kuzirian M.S., Snyder L.M., Matsushita M., Lee M.C., Ferguson C., Homanics G.E., Barth A.L, & Ross S.E. (2015). Generation of a KOR-Cre Knockin Mouse Strain to Study Cells Involved in Kappa Opioid Signaling. Genesis (New York, N.Y. : 2000), 54(1), 29-37.

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Multimarker Immunostaining of Neuronal and Glial Cells

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Neurons were immunostained with a mouse monoclonal anti-NeuN antibody (1:200, MilliporeSigma, Carlsbad, CA, United States), astrocytes were detected by means of a polyclonal rabbit antibody anti-Glial Fibrillary Acidic Protein (GFAP, 1:500, DakoCytomation, Glostrup, Denmark), Cytochrome C with a mouse monoclonal antibody (1:200, Abcam, Cambridge, United Kingdom). Activated mTOR was detected using a polyclonal rabbit primary antibody raised against phospho-(Ser2448)-mTOR (1:100, Abcam, Cambridge, United Kingdom). Fluorescent secondary antibodies: Alexa Fluor 488 donkey anti rabbit (fluorescence in green, 1:400), Alexa Fluor 555 donkey anti mouse (fluorescence in red, 1:400), Alexa Fluor 635 goat anti-rabbit (fluorescence in far red, 1:400) (all from Life Technologies, Carlsbad, CA, United States). All primary and secondary antibodies were dissolved in Blocking Buffer (BB, 10% Normal Goat Serum, 0.05% NaN₃ in PBS-TX). All procedures were carried out with the free-floating method in wells of a 24-well plate (Cerbai et al., 2012 (link); Lana et al., 2013 (link)).

Fusco I., Ugolini F., Lana D., Coppi E., Dettori I., Gaviano L., Nosi D., Cherchi F., Pedata F., Giovannini M.G, & Pugliese A.M. (2018). The Selective Antagonism of Adenosine A2B Receptors Reduces the Synaptic Failure and Neuronal Death Induced by Oxygen and Glucose Deprivation in Rat CA1 Hippocampus in Vitro. Frontiers in Pharmacology, 9, 399.

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Immunofluorescence Localization of PMCA1

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HOB cells were cultured in 8-well glass chambers (AGC Techno Glass Co.,Ltd., Shizuoka, Japan) for 1 day. Cells were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) and washed with 1× PBS (Thermo Fisher Scientific K.K., Tokyo, Japan). After 10 min of incubation with blocking buffer (Nacalai Tesque, Kyoto, Japan) at room temperature, mouse monoclonal anti-PMCA1 (Santa Cruz Biotechnology, Dallas, Texas, USA; sc-398413, F-10, 1:200) was applied for 6 h to detect human PMCA1. Secondary antibody (Alexa Fluor^® 555 donkey anti-mouse; Thermo Fisher Scientific K.K.) was then applied for 1 h. Stained samples were mounted in mounting medium containing 4,6-diamidino- 2-phenylindole (Abcam, Cambridge, UK). Immunostained samples were analyzed and observed using a fluorescence microscope (BZ9000; KEYENCE Co., Osaka, Japan). For negative control, the cells were incubated with nonimmune antibody diluted to equivalent concentrations to that of the primary antibody (data not shown).

Kimura M., Mochizuki H., Satou R., Iwasaki M., Kokubu E., Kono K., Nomura S., Sakurai T., Kuroda H, & Shibukawa Y. (2021). Plasma Membrane Ca2+–ATPase in Rat and Human Odontoblasts Mediates Dentin Mineralization. Biomolecules, 11(7), 1010.

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Immunofluorescence Staining of Cultured Cells

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Antibody staining of cells was performed in the same way as described in siRNA Screening and Transfection. Plates were washed once with PBS and fixed with 4% paraformaldehyde incubated for 10 min at room temperature. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature, incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) or anti-cleaved caspase 3 (Asp175) (Cell Signaling catalog no. 9661) diluted to 1 µg/mL or according to manufacturer’s instructions in blocking buffer. Plates were then washed three times with PBS, stained with the secondary antibodies Alexa Fluor 555 donkey anti-mouse (Thermo Fisher Scientific) and/or Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific) at 1 µg/mL, the nuclear dye DRAQ5 (abcam) at 10 µM, and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) at 12.6 nM in blocking buffer. Secondary stains were incubated for 2 h at room temperature protected from light, and plates were finally washed three times with PBS before being imaged using the Perkin-Elmer Operetta High-Content Imaging System.

Vietri Rudan M., Mishra A., Klose C., Eggert U.S, & Watt F.M. (2020). Human epidermal stem cell differentiation is modulated by specific lipid subspecies. Proceedings of the National Academy of Sciences of the United States of America, 117(36), 22173-22182.

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