A21052

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A21052 is a laboratory instrument designed for conducting sample analysis and processing. It is a core component of various analytical workflows across various industries and research applications. The product specification and technical details are available upon request.

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Lab products found in correlation

18 protocols using a21052

Comprehensive Cell Cytoskeleton Staining

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After the end of the experiment, the scaffolds were fixed in 3% paraformaldehyde (Thermo Fisher Scientific) for 15 min and permeabilized in 1% Triton X-100 (Sigma-Aldrich) for 20 min. The actin cytoskeleton and cell nuclei were stained with Alexa Fluor 488 phalloidin (1:1000, A12379, Thermo Fisher Scientific) and DAPI (2 μg/ml, D1306, Invitrogen), respectively. Col-I primary antibody (ab90395, Abcam plc) was added to the medium at a dilution of 1:200 30 min before fixation to facilitate ECM staining, as described previously (44 (link)), and counterstained with AAlexa Fluor 633 goat anti-mouse secondary antibody (A-21052, Thermo Fisher Scientific) at 1:50. YAP was stained with YAP (63.7) primary mouse monoclonal antibody (sc-101199, Santa Cruz Biotechnology) at 1:200 and counterstained with Alexa Fluor 633 goat anti-mouse secondary antibody (A-21052, Thermo Fisher Scientific) at 1:50. α-SMA was stained with primary mouse anti–α-SMA antibody [0.N.5] (ab18147, Abcam plc) at 1:100 and counterstained with Alexa Fluor 546 goat anti-mouse secondary antibody (A-11030, Thermo Fisher Scientific) at 1:100. After washing with phosphate-buffered saline (PBS), primary and secondary antibodies were applied for 30 min each except for Col-I primary antibody, as mentioned above.

Kollmannsberger P., Bidan C.M., Dunlop J.W., Fratzl P, & Vogel V. (2018). Tensile forces drive a reversible fibroblast-to-myofibroblast transition during tissue growth in engineered clefts. Science Advances, 4(1), eaao4881.

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Immunostaining of Drosophila CNS and PNS

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For CNS immunostaining, whole flies immobilized with insect pin on abdomen were fixed in 4% PFA for 2 hours on nutator at room temperature. After three 1 min wash in PBT, flies were dissected in PBS buffer to get the whole CNS. CNS samples were further washed by three times in 1 min PBT and then blocked for 30 min in 4% NGS. Staining with primary antibody was performed in 4°C overnight on nutator. Samples were then washed 3 times for 20 min in PBT. Secondary antibody incubation was performed for 2 hours in room temperature. Samples were washed again in PBT for 3 times; mounted in VectaShield for imaging. PNS dissection and eye bristles immunostaining was performed using the published protocol [10 (link)]. In short, whole flies were washed in 100% ethanol and then PBS, specific body parts were then pulled and mounted in VectaShield on microscope slides for imaging. The following primary antibodies were used: chicken polyclonal to GFP (Abcam 13970, 1:500) and mouse monoclonal brp antibody (DSHB nc82, 1:200). The secondary antibodies were anti-chicken Alexa Fluor 488 (Invitrogen Molecular Probes A-11039, 1:500) and anti-mouse Alexa Fluor 633 (Invitrogen Molecular Probes A-21052, 1:500). Confocal images were taken on a Zeiss LSM710 microscope. Images were then processed in ImageJ.

Zhang N., Guo L, & Simpson J.H. (2020). Spatial Comparisons of Mechanosensory Information Govern the Grooming Sequence in Drosophila. Current biology : CB, 30(6), 988-1001.e4.

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Immunostaining of Drosophila CNS and PNS

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Zhang N., Guo L, & Simpson J.H. (2020). Spatial Comparisons of Mechanosensory Information Govern the Grooming Sequence in Drosophila. Current biology : CB, 30(6), 988-1001.e4.

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Immunostaining Protocol for Drosophila Brains

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Immunostaining was performed as previously described in [11 (link)]. Briefly, fly heads were fully dissected and fixed in 400 µL of 4% paraformaldehyde (PFA), 0.3% Triton X-100 for 20 min on ice. Once fixation was completed, three quick washes followed by three 20-min washes were performed in PBS, 0.3% Triton X-100. Blocking was achieved with 1 h incubation in 5% normal goat serum (NGS), PBS, 0.3% Triton X-100 at room temperature on a rocking platform. Brains were incubated with the primary antibodies for two nights at 4°C on a rocking platform. Brains were then washed and incubated with the secondary antibodies overnight at 4°C. Vectashield (Vector Laboratories) was used as slide mounting medium. Antibodies used in this study and dilutions were as follows: Mouse anti-nc82 (mouse, Developmental Studies Hybridoma Bank, 1:100), Rabbit anti-GFP (G10362, Invitrogen. 1:500), Goat anti-Rabbit IgG Alexa Fluor 488 1:200 (A11008, Thermo Fisher, 1:200) and Goat anti-mouse IgG-Alexa Fluor 633 1:200 (mouse, A21052, Thermo Fisher, 1:200).

Stickley L., Koch R, & Nagoshi E. (2023). The utility and caveat of split-GAL4s in the study of neurodegeneration. Fly, 17(1), 2192847.

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Multicolor Immunostaining of Isolated Islets

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Immunostaining was conducted on isolated islets, fixed in 4% paraformaldehyde before being permeabilised using 0.1% Triton X-100 (Sigma). After blocking with 5% goat serum, islets were incubated overnight (4°C) with primary antibodies before incubation with secondary antibodies. Fluorescent staining was visualised using a laser-scanning confocal microscopy (BioRad) controlled by LaserSharp2000 (BioRad).
Antibodies used in this study were: rabbit FITC 495-conjugated anti-GFP (1:250; DS-PB-00926; InsightBio, Wembley, United Kingdom), mouse anti-glucagon (1:1000; G2654; Sigma), guinea-pig anti-insulin (1:400; A0564; Dako, Santa Clara, California, United States), goat anti-somatostatin (1:100; sc-7819; Santa Cruz Biotechnology, Dallas, Texas, United States); Alexa Fluor 633 goat anti-mouse IgG (1:500; A-21052; ThermoFisher); Alexa Fluor 594 goat anti-guinea pig IgG (1:500; A-11076; ThermoFisher); and Alexa Fluor 546 donkey anti-goat IgG (1:100; A-11056; ThermoFisher).

Acreman S., Ma J., Denwood G., Gao R., Tarasov A., Rorsman P, & Zhang Q. (2024). The endoplasmic reticulum plays a key role in α-cell intracellular Ca2+ dynamics and glucose-regulated glucagon secretion in mouse islets. iScience, 27(5), 109665.

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Microscopic Visualization of Mitochondria and Cytoskeleton

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HeLa cells seeded on 35 mm glass-bottom dishes were fixed in 3.7% paraformaldehyde at 37 °C for 12 min, washed in PBS × 3 and permeabilized in 0.2% Triton X-100 in PBS for no longer than 5 min at room temperature. Cells were again washed in PBS × 3 and blocked in 0.5% BSA in PBS for 10 min at room temperature. Cells were then incubated for 30 min with Phalloidin-488 (1:200; A12379, ThermoFisher), anti-Tom20 rabbit polyclonal antibody (1:50; sc-11415, Santa Cruz) and/or anti-DRP1 mouse monoclonal antibody (1:100; ab56788, Abcam) in PBS with 0.5% BSA. Cells were then washed in PBS × 3 and incubated with either AlexaFluor goat-anti-rabbit-594 (1:300; A-11037, ThermoFisher) or AlexaFluor goat-anti-rabbit 555 (1:300; A21429, ThermoFisher), AlexaFluor goat-anti-mouse-647 (1:300; A21052, ThermoFisher) and Hoechst dye (62249; ThermoFisher). Cells were washed in PBS × 3 and imaged.

Moore A.S., Wong Y.C., Simpson C.L, & Holzbaur E.L. (2016). Dynamic actin cycling through mitochondrial subpopulations locally regulates the fission–fusion balance within mitochondrial networks. Nature Communications, 7, 12886.

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EdU Labeling and Immunostaining in Tissue

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Animals were treated with 50 mg/kg EdU i.p. (BCK488-IV-IM-S, baseclick GmbH) 4 h prior to euthanasia. Animals were cardially perfused with 4.5% phosphate-buffered formaldehyde solution (Roti-Histofix 4.5%, 2213, Carl Roth). Tissue was frozen after cryoprotection with 30% sucrose solution and 10 µm cryosections were cut. The sections were permeabilized with 0.5% Triton X-100 in PBS for 20 min. The EdU Click Kit (BCK488-IV-IM-S, baseclick GmbH) was used for EdU detection. After washing, sections were incubated with the reaction cocktail for 30 min as per the manufacturer’s instructions. Afterwards sections were incubated with anti-nestin (1:400, ab6320, Abcam, RRID:AB_308832) and anti-CD31 (1:50; PA5-16301, ThermoFisher Scientific, RRID:AB_10981955) antibodies.Goat anti-rabbit Alexa Fluor 594 (1:400; A-11037, ThermoFisher Scientific, RRID:AB_2534095) and goat anti-mouse Alexa Fluor 633 (1:400; A-21052, ThermoFisher Scientific, RRID:AB_2535719) secondary antibodies were used. Images were acquired using a Leica TCS SP5 confocal microscope (LAS software version 2.7.3.9723).

Jung E., Osswald M., Ratliff M., Dogan H., Xie R., Weil S., Hoffmann D.C., Kurz F.T., Kessler T., Heiland S., von Deimling A., Sahm F., Wick W, & Winkler F. (2021). Tumor cell plasticity, heterogeneity, and resistance in crucial microenvironmental niches in glioma. Nature Communications, 12, 1014.

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Immunohistochemistry of c-Fos in Mouse Brain

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Mice were deeply anesthetized with Euthasol and transcardially perfused with 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4. Coronal brain slices (35 μm) were prepared, and a standard immunohistochemistry protocol was followed (17 (link)). The primary antibody used was rabbit anti–c-Fos (1:1000; Abcam, ab-222699 RRID: AB_2891049). Alexa Fluor secondary antibodies (633-goat anti-rabbit; 1:1000; Life Technologies, A21052, RRID: AB_2535719) were used to visualize the signal, and images were acquired by confocal microscopy (Zeiss, LSM880) (17 (link)).

Huang Z., Liu L., Zhang J., Conde K., Phansalkar J., Li Z., Yao L., Xu Z., Wang W., Zhou J., Bi G., Wu F., Seeley R.J., Scott M.M., Zhan C., Pang Z.P, & Liu J. (2022). Glucose-sensing glucagon-like peptide-1 receptor neurons in the dorsomedial hypothalamus regulate glucose metabolism. Science Advances, 8(23), eabn5345.

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Fluorescent Labeling of Hypothalamic Neurons

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The CRH-Ires-Cre mice were injected with AAV-DIO-mCherry. Two weeks after, mice were sacrificed and coronal hypothalamic slices (300 μm) were incubated at 30°C for 20 min in oxygenated artificial cerebrospinal fluid (ACSF) containing (in mM): 125 NaCl, 2.5 KCl,1.25 NaH₂PO₄, 25 NaHCO₃, 2.5 Glucose, 22.5 Sucrose, 2.5 CaCl₂, 1.2 MgCl₂. The slices were incubated with 100 nM Fluorescein-Trp²⁵- Exendin-4 (F-Exn4, Anaspec Inc., cat # AS-63899) in ACSF at room temperature for 15 min. 4% PFA was used to fix the tissue directly after removing ACSF. The slices were washed well with PBS prior to immunostaining. The primary antibodies used were: anti-OXT (1:1000, Millipore, ab911); anti-AVP (1:1000, Millipore, ab1565). The secondary antibodies used were: Alex-546-goat anti-rabbit (1:1000, Life Technologies, A11035) and Alex-633-goat anti-mouse (1:1000, Life Technologies, A21052).

Liu J., Conde K., Zhang P., Lilascharoen V., Xu Z., Lim B.K., Seeley R.J., Zhu J.J., Scott M.M, & Pang Z.P. (2017). Enhanced AMPA receptor trafficking mediates the anorexigenic effect of endogenous glucagon like peptide-1 in the paraventricular hypothalamus. Neuron, 96(4), 897-909.e5.

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Immunofluorescence Microscopy of Lipid Regulators

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MCF10CA1a cells were cultured in a #1.5H-N high performance glass bottom 12 well plate (Cellvis) and processed for immunofluorescence microscopy. The cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with BlockAid (Invitrogen, B10710). Cells were probed with antibodies for PLIN3, SQLE, and NSDHL (Sigma, HPA006427; SantaCruz Biotechnologies, sc-271651; Atlas Antibodies, HPA000571, respectively). Proteins were detected using secondary AlexaFluor antibodies (Life Technologies, A-21070 and A-21052), and cells were counterstained for neutral lipids using 1 μg/mL 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503; Life Technologies, Grand Island, NY, United States), and for nuclei using 300 nM 4’,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI; Invitrogen, D1306). Samples were imaged using a Nikon A1R-MP inverted confocal microscope (Nikon Instruments Inc., Melville, NY, United States). Images were acquired using the Plan Apo λ 100x Oil objective, 76.63 µm pinhole size, and DAPI, FITC, and Cy5 lasers. All image processing was conducted using Nikon NIS-Elements AR acquisition and analysis software. A Landweber 2D deconvolution algorithm was implemented, with point scan confocal modality, clear noise, and 12, 12, 12 iterations.

Zembroski A.S., Andolino C., Buhman K.K, & Teegarden D. (2021). Proteomic Characterization of Cytoplasmic Lipid Droplets in Human Metastatic Breast Cancer Cells. Frontiers in Oncology, 11, 576326.

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