Donkey anti mouse 488

Manufactured by Thermo Fisher Scientific

Sourced in United States

Donkey anti-mouse 488 is a secondary antibody conjugated with the fluorescent dye Alexa Fluor 488. It is designed to detect and visualize primary antibodies raised in mouse.

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Close spelling variants of this product

AF 488 donkey anti-mouse Donkey anti-rabbit and anti-mouse Alexa Fluor 488 antibodies AlexaFluor 488-conjugated polyclonal donkey anti-mouse IgG Alexa Fluor 488 donkey anti-mouse IgG (H+L) Donkey anti-mouse IgG secondary antibody conjugated to Alexa Fluor 488 Alexa Fluor-conjugated donkey anti-rabbit (488) or donkey anti-mouse (546) antibodies Alexa Fluor 488-Donkey anti-mouse (A32766) Donkey anti-mouse AlexaFluor 488 secondary antibody Donkey anti-mouse IgG-conjugated Alexa Fluor 488 or 594 Alexa Fluor 488 donkey anti-mouse secondary antibody

33 protocols using donkey anti mouse 488

Immunocytochemistry for Myosin Heavy Chain

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Following functional analysis, cells were fixed in a 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) solution for 15 min. Cells were rinsed three times with PBS and then permeabilized in a PBS solution containing 0.1% Triton X-100 for 20 min. Cells were then incubated in a blocking buffer (5% donkey serum with 0.1% bovine serum albumin in PBS) for 20 min. Cells were incubated overnight at 4°C in a primary antibody solution containing the blocking buffer and an antibody against MyHC all classes (DSHB A4.1025) at a 1:10 dilution. On the following day, the primary antibody solution was removed and the cells were rinsed with 3 × 10-min washes in PBS. Cells were then incubated with a secondary antibody solution for 2 hr. This solution contained blocking buffer, donkey-anti-mouse-488 (Invitrogen) diluted 1:250, and phalloidin 568 at 1:40 dilution (Invitrogen). The phalloidin was used for actin filament visualization. After this incubation, cells were rinsed three times with PBS, with 10 min in between each rinse. The second rinse contained a 4′, 6-diamidino-2-phenylindole (DAPI) solution (1:10,000 in PBS; Life Technologies, New York, NY) for visualization of nuclei.

Najjar S.A., Smith A.S., Long C.J., McAleer C.W., Cai Y., Srinivasan B., Martin C., Vandenburgh H.H, & Hickman J.J. (2019). A multiplexed in vitro assay system for evaluating human skeletal muscle functionality in response to drug treatment. Biotechnology and bioengineering, 117(3), 736-747.

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Immunofluorescent Staining of iPSC-derived Cardiomyocytes

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At day 21 of differentiation, human iPSC-derived cardiomyocytes on 13 mm tissue-culture treated coverslips were rinsed with PBS and treated with 100 nM Mitotracker Far Red (Invitrogen P36970) for 35 min at 37°C. Coverslips were fixed in 4% Paraformaldehyde (Electron Microscopy Sciences, 15710) at 4°C for 15 min, rinsed and stored in PBS. Cells were permeabilized with 0.5% Triton-X100 (Millipore 648466) for 10 min at room temperature, rinsed in PBS, blocked in 3% Bovine-Serum Albumin in PBS-Tween 20 0.1% for 90 min at room temperature, and rinsed in PBS. They were incubated overnight at 4 °C in 1:500 Mouse-anti-α-Actinin (Sigma A7811) and 1:300 Rabbit-anti-Pan-Cadherin (Applied Biosystems AB16505) in 3% BSA in PBS-Tween 0.1% at 4°C overnight. Coverslips were washed on a rocker at room temperature 3X for 5 min in PBS-Tween 0.1%, incubated for 1 hour at room temperature in secondary antibodies of 1:400 Donkey-anti-Mouse-488 (Invitrogen A21202) and 1:400 Donkey-anti-Rabbit-568 (Invitrogen A10042), and washed 3X for 5 min at RT on a rocker in PBS-Tween 0.1%. Cells were then stained in 1 μg/ml DAPI for 10 min at room temperature. Coverslips were mounted in Prolong Diamond (Invitrogen P36970) under a #1.5 coverslip. Imaging was done on a Nikon A1R with ×20 objectives.

Wang J., Morgan W., Saini A., Liu T., Lough J, & Han L. (2022). Single-cell transcriptomic profiling reveals specific maturation signatures in human cardiomyocytes derived from LMNB2-inactivated induced pluripotent stem cells. Frontiers in Cell and Developmental Biology, 10, 895162.

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Co-localization of p53, Rb, and E7

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The primary antibodies used for co-localization studies included: a rabbit polyclonal antibody to p53 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-6243); a rabbit polyclonal antibody to Rb (Santa Cruz Biotechnology, Santa Cruz, CA, sc-50); and a mouse monoclonal antibody to E7 (Santa Cruz Biotechnology, Santa Cruz, CA, sc-65711). Secondary antibodies used included: donkey anti-mouse 488 and donkey anti-rabbit 568 (obtained from Invitrogen, Waltham, MA). DAPI was used as a nuclear fluorescent stain that binds strongly to A-T rich regions in DNA. Confocal images were obtained with an Olympus Fluoview 100 confocal microscope and companion software FV10-ASW1.6. Images were acquired at a resolution of 1024 × 1024 pixels.

Zhang X., Hou W., Epperly M.W., Rigatti L., Wang H., Franicola D., Sivanathan A, & Greenberger J.S. (2016). Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2−/− mouse long-term bone marrow cultures. Oncotarget, 7(42), 68449-68472.

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Antibody Staining for Extracellular Matrix

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The following antibodies were used: rabbit anti-mouse fibronectin (Sigma, St. Loius, MO, USA, Cat# F3648), goat anti-mouse integrin α8 (R&D Systems, Minneapolis, MN, USA, Cat# AF4076), mouse anti-bovine laminin β2 C4 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), rabbit anti-mouse collagen IVα5 (Cosgrove) and rabbit anti-human laminin α2 (gift from Dr. Peter Yurchenco, Robert Wood Johnson Medical School, Piscataway, NJ, USA). Alexa-fluor conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) included: donkey anti-rabbit 594 for anti-fibronectin and anti-collagen IVα5, and donkey anti-goat 568 for anti-integrin α8 and donkey anti-rabbit 488 for anti-laminin α2 or donkey anti-mouse 488 for anti-laminin β2 C4 (for dual staining). Negative controls were performed using the host serum in combination with the specific antibodies described above (S1 Fig).

Clark S.D., Nabity M.B., Cianciolo R.E., Dufek B, & Cosgrove D. (2016). X-Linked Alport Dogs Demonstrate Mesangial Filopodial Invasion of the Capillary Tuft as an Early Event in Glomerular Damage. PLoS ONE, 11(12), e0168343.

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SDS-PAGE Analysis of Cellular Protein Expression

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For sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) cell lysates of CG4, CG4_venus, and CG4_wt-α-syn cells (n = 3 each) were extracted using RIPA buffer and 15 μg of proteins were loaded on a 4-12 % Novex® NuPAGE Bis-Tris-Mini gels (Invitrogen) and afterwards transferred on a polyvinylidene fluoride membrane (Immobilon PVDF-FL, 0.45 μm; Millipore). Membranes were blocked (PBS containing 0.1 % Tween-20 and 1 % BSA) for 1 h at RT and incubated in primary antibodies diluted in 0.1 % sodium azide, 0.1 % Tween 20, and 1 % BSA in PBS overnight at 4°C: monoclonal rat anti-MBP (1:200; MCA409S; AbD Serotec), monoclonal mouse anti-GAPDH (1:100.000; clone GB-69; Sigma), and monoclonal anti-CNPase (1:1000; clone 11-5B; Millipore) were detected with respective secondary antibodies diluted in blocking buffer (1 h at RT): donkey anti-mouse 488 (1:3000), donkey anti-rat 488 (1:3000), and donkey anti-mouse 647 (1:1000; all Invitrogen). Fluorescent signals were captured using a Fusion FX7 detection system (Peqlab). Results were quantified using Bio1D software (Vilber Lourmat) and normalized to GAPDH.

May V.E., Ettle B., Poehler A.M., Nuber S., Ubhi K., Rockenstein E., Winner B., Wegner M., Masliah E, & Winkler J. (2014). α-Synuclein impairs oligodendrocyte progenitor maturation in multiple system atrophy. Neurobiology of aging, 35(10), 2357-2368.

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Immunofluorescence Analysis of HIF-1α

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Cells were seeded onto glass cover slides and allowed to attach for 24 h. Medium was removed and cells fixed with 4% paraformaldehyde for 10 min. After fixation, cells were washed with PBS and permeabilized with 0.2% Triton^®-X-100 (Serva, Heidelberg, Germany) for 5 min. Subsequently, cells were incubated in StartingBlock^TM (PBS) blocking buffer (ThermoFisher Scientific, Waltham, MA, USA) for 1 h at room temperature. The following primary antibodies were added for 1 h at room temperature: rabbit anti HIF-1α (dilution 1:50; EP1215Y, Abcam, Cambridge, UK) and chicken anti cytokeratin 8/18 (dilution 1:100; Sigma-Aldrich). After removal of primary antibodies, cover slides were washed with PBS and secondary antibodies added: goat anti rabbit 555 (1:200, Invitrogen) and donkey anti mouse 488 (1:200, Invitrogen). Subsequently, slides were mounted in Vectashield mounting medium containing DAPI (Vector Laboratories, Burlington, CA, USA). Cells were visualized using fluorescent microscopy on a Zeiss Axio Imager Z2. HIF-1α staining intensity was scored using TissueQuest software (TissueGnostics, Vienna, Austria) and normalized to DAPI positive cells.

Weber A., Klocker H., Oberacher H., Gnaiger E., Neuwirt H., Sampson N, & Eder I.E. (2018). Succinate Accumulation Is Associated with a Shift of Mitochondrial Respiratory Control and HIF-1α Upregulation in PTEN Negative Prostate Cancer Cells. International Journal of Molecular Sciences, 19(7), 2129.

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Immunohistochemical Detection of Glut1 in EAE

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We used immunostaining to detect the glucose transporter 1 (Glut1). Optically cleared spinal columns (n = 6 control, n = 5 EAE) were rehydrated in decreasing methanol/H₂O series (100%, 80%, 60%, 40%, 20%, 1 h each) and finally washed in 1 × PBS overnight, in order to partially reverse the clearing. Then, the lumbar region of the spinal cord was dissected out from the vertebral column and sectioned with a vibratome (100 µm thickness). Selected coronal lumbar spinal cord sections were histochemically stained. Briefly, a blocking solution (0.3% Triton X-100, 5% BSA, and 1% serum) was applied for one hour at room temperature (RT) to minimize unspecific antibody binding. Then, the primary antibody anti-Glut1 (MABS132, 1:250) was allowed to label the tissue overnight at 4 ◦C under gentle shaking. The next day, sections were washed three times with 1 × PBS before incubating with the secondary antibody (Donkey Anti-mouse 488, Invitrogen, 1:500) for 90 min at RT under gentle shaking. Following that, three washes with 1 × PBS, 40,6-diamidin-2-fenilindolo (DAPI, 1:1000) were performed before the sections were mounted.

Ramos-Vega M., Kjellman P., Todorov M.I., Kylkilahti T.M., Bäckström B.T., Ertürk A., Madsen C.D, & Lundgaard I. (2022). Mapping of neuroinflammation-induced hypoxia in the spinal cord using optoacoustic imaging. Acta Neuropathologica Communications, 10, 51.

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Fixation and Fluorescent Antibody Staining of Drosophila Embryos

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Standard protocols were followed for fixation of 2–4 hr embryos in
37% formaldehyde solution (Kosman et al.,
2004). Fluorescent antibody staining was performed according to
standard protocols (Kosman et al., 2004 (link)),
with primary antibodies against Dl (1:10 dilution; DSHB anti-dorsal 7A4) and
histone (1:5000 dilution; abcam ab1791). The anti-Dl monoclonal antibody (DSHB
Hybridoma Product anti-dorsal 7A4) was deposited to the DSHB by R. Steward
(Whalen and Steward, 1993 (link)), and was
obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of
the NIH and maintained at The University of Iowa, Department of Biology, Iowa
City, IA 52242. Secondary antibodies used were donkey anti-mouse-488 (1:500
dilution; Invitrogen A21202) and donkey anti-rabbit-546 (1:500 dilution;
Invitrogen A10040),

Schloop A.E., Carrell-Noel S., Friedman J., Thomas A, & Reeves G.T. (2020). Mechanism and implications of morphogen shuttling: lessons learned from Dorsal and Cactus in Drosophila. Developmental biology, 461(1), 13-18.

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Multicolor Immunohistochemistry of Brain Tissue

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Brian tissue sections were incubated overnight with primary antibodies at 4°C and then with corresponding fluorochrome-conjugated secondary antibodies at room temperature for 1 h. The following primary antibodies were used: anti-human NKp46 (195314; R&D Systems), CD49a (SR84; BD Bioscience), CD57 (MA1-81071; Invitrogen), CD68 (ED1; Abcam), CD4 (H-370; Santa Cruz), CD8 (UCH-T4; Santa Cruz), CD19 (HIB19; BioLegend), CD16b (CLB-gran11.5; BD Bioscience), CD66b (polyclonal; Bioss), perforin (dG9; BioLegend), CD69 (D-3; Santa Cruz), Caspase-3 (9661; CST), CD31 (JC/70A; Abcam); anti-mouse NKp46 (M20; Santa Cruz), CD49a (Ha31/8; BD Bioscience), ly6G (1A8; BioLegend), TMEM119 (28-3; Abcam), CD68 (ED1; Abcam), CD8 (53-6.7; eBioscience), CD4 (GK1.5; eBioscience), CD19 (1D3; BD Bioscience), CD31 (polyclonal; Abcam), claudin5 (4C3C2; Invitrogen), and ZO-1 (ZO-1-1A12; Invitrogen). The following fluorochrome-conjugated secondary antibodies were used: donkey anti-rabbit 488 (1:1,000; Invitrogen), donkey anti-rabbit 546 (1:1,000; Invitrogen), donkey anti-goat 546 (1:1,000; Invitrogen), donkey anti-mouse 594 (1:1,000; Invitrogen), donkey anti-mouse 488 (1:1,000; Invitrogen), goat anti-rat 488 (1:1,000; Invitrogen), and goat anti-rat 555 (1:1,000; Invitrogen). Images were acquired on a fluorescence microscope (Olympus BX-61).

Li Z., Li M., Shi S.X., Yao N., Cheng X., Guo A., Zhu Z., Zhang X, & Liu Q. (2020). Brain transforms natural killer cells that exacerbate brain edema after intracerebral hemorrhage. The Journal of Experimental Medicine, 217(12), e20200213.

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Immunofluorescence Staining of PIAS1

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Cells were seeded onto glass coverslips and allowed to attach for 24 h. Depending on the assay, they were grown for 2 d without treatment (for localization studies) or transfected with siRNA against PIAS1 or control siRNA (for siRNA efficiency studies and p21 expression experiments). Subsequently, the cells were again washed with PBS and fixed with 4% paraformaldehyde for 10 min. The cells were washed with PBS and permeabilized with PBS+1% bovine serum albumin+0.2% Triton X100 for 5 min. After 30 min blocking with PBS+1% bovine serum albumin, coverslips were incubated for 1 h with primary antibodies (PIAS1 [D33A7] XP^® Rabbit mAb 1:50, Cell Signaling, Frankfurt am Main, Germany). After washing with TBS + 0.1% Tween20, coverslips were incubated with the following fluorescence-labeled secondary antibodies: goat anti-rabbit 555 and donkey anti-mouse 488 (all Invitrogen). Coverslips were finally washed with TBS and mounted with Vectashield Hard Set mounting medium containing DAPI (Vector Laboratories, Burlingame, CA) on glass slides. The cells were visualized using fluorescent microscopy on an Axio Observer Z1 microscope (Carls Zeiss AG, Overkochen, Germany).

Erb H.H., Ebert M., Kuhn R., Donix L., Haferkamp A., Seed R.I, & Jüngel E. (2019). PIAS1 is not suitable as a urothelial carcinoma biomarker protein and pharmacological target. PLoS ONE, 14(10), e0224085.

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