A21202

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, China, France

The A21202 is a laboratory instrument designed for the analysis and measurement of various samples. It provides accurate and reliable data to support scientific research and industrial applications. The core function of this product is to perform precise quantitative and qualitative analysis, but a detailed description without interpretation or extrapolation is not available.

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Lab products found in correlation

228 protocols using a21202

Macrophage Infiltration and Inflammatory Markers in Sciatic Nerve Injury

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The recovered control, sham and CCI sciatic nerves were prepared for immunohistochemical examination using mouse anti rat CD68 antibody (MCA341R, AbD Serotech, Raleigh, NC) and Alexa fluor 488 donkey anti-mouse secondary antibody (A-21202, Invitrogen, Carlsbad, CA) to assess the presence of macrophages that infiltrate the nerve. The double immunofluorescence studies were also performed to reveal the expression of COX-2 and PGE2 in relationship to the CD68 positive infiltrating macrophages using, rabbit anti-COX-2, 1:100 (ab15191, AbD Serotech) and rabbit anti-prostaglandin E2 antibody, 1:100 (ab2318, AbD Serotech) along with mouse CD68, 1:100 (MCA341R, AbD Serotech) in treated and untreated rat groups. The secondary antibodies used were Alexa fluor 488 donkey anti-mouse, 1:200 (A-21202, Invitrogen), Alexa fluor 546 donkey anti-goat, 1:200 (A-11056, Invitrogen) and Alexa fluor 647 donkey anti-rabbit, 1:200 (A-31573, Invitrogen). All the antibody dilutions were prepared using 1:20 normal donkey serum in 1× PBS, pH 7.4. Antigen retrieval was performed during double immunofluorescence with COX-2 and CD68 primary antibodies, using sodium citrate buffer (10mM sodium citrate, 0.1% Tween 20, pH 8.5). Antigen retrieval was not required during double immunofluorescence with PGE2 and CD68 primary antibodies.

Janjic J.M., Vasudeva K., Saleem M., Stevens A., Liu L., Patel S, & Pollock J.A. (2018). Low-dose NSAIDs reduce pain via macrophage targeted nanoemulsion delivery to neuroinflammation of the sciatic nerve in rat. Journal of neuroimmunology, 318, 72-79.

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Comprehensive Antibody Labeling Protocol

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The following primary antibodies were used: Guinea pig anti-CAPS2 [1:100 (Sadakata et al., 2004 (link); Sadakata et al., 2006 (link))], rabbit anti-α-amylase (1:200; A8273, Sigma-Aldrich, Saint Louis, MO, United States), mouse anti-actin, α-smooth muscle-Cy3 (1:500; C6198, Sigma-Aldrich), goat anti-calnexin (1:100; sc-6465, Santa Cruz Biotechnology, Santa Cruz, CA, United States), mouse anti-GM130 (1:150; 610822, BD Transduction Laboratories, San Jose, CA, United States), mouse APC anti-mouse CD3ε (1:300; 100312, BioLegend Inc., San Diego, CA, United States), and rabbit anti-TGN38 (1:100; T9826, Sigma-Aldrich). The secondary antibodies used were: Alexa Flour 488 goat anti-guinea pig IgG (H + L) (1:500; A11073, Invitrogen, Carlsbad, CA, United States), Alexa Flour 488 goat anti-mouse IgG (H + L) (1:1,000; A28175, Invitrogen), Alexa Flour 488 donkey anti-rabbit IgG (H + L) (1:5,000; A21206, Invitrogen), Alexa Flour 488 donkey anti-mouse IgG (H + L) (1:1,000; A21202, Invitrogen), Alexa Flour 488 donkey anti-mouse IgG (H + L) (1:1,000; A21202, Invitrogen), Alexa Flour 546 donkey anti-goat IgG (H + L) (1:1,000; A11058, Invitrogen), Alexa Fluro555 donkey anti-goat IgG (H + L) (1:1,000; A11055, Invitrogen), and Alexa Flour 594 donkey anti-goat IgG (H + L) (1:1,000; A11058, Invitrogen).

Sato Y., Tsuyusaki M., Takahashi-Iwanaga H., Fujisawa R., Masamune A., Hamada S., Matsumoto R., Tanaka Y., Kakuta Y., Yamaguchi-Kabata Y., Furuse T., Wakana S., Shimura T., Kobayashi R., Shinoda Y., Goitsuka R., Maezawa S., Sadakata T., Sano Y, & Furuichi T. (2022). Loss of CAPS2/Cadps2 leads to exocrine pancreatic cell injury and intracellular accumulation of secretory granules in mice. Frontiers in Molecular Biosciences, 9, 1040237.

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Quantifying Viral Protein Expression

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A549 cells were infected at an empirically determined MOI (by titration) that gave ~10% levels of infection for 6 hr, after which cells were fixed, permeablised and stained for a range of viral proteins. M2 was detected by ab5416 (Abcam, Cambridge, UK), NS1 by an in-house (rabbit) antiserum, NA by an in house (mouse) antiserum, NP by ab20343 (Abcam, Cambridge, UK) and PB2 by an in house rabbit antiserum. Secondary antibodies were species specific Alexafluor-488 or Alexafluor-546 (A21202, A11005, A21207 and A21206, Life Technologies, Loughborough, UK).

Gaunt E., Wise H.M., Zhang H., Lee L.N., Atkinson N.J., Nicol M.Q., Highton A.J., Klenerman P., Beard P.M., Dutia B.M., Digard P, & Simmonds P. (2016). Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection. eLife, 5, e12735.

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Rac1 Activation in B16F10 Cells

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Following various pretreatments, the B16F10 cells were fixed with 4% paraformaldehyde (PFA), and then permeabilized with 0.1% Triton X-100. Rac1 antibody (1∶100) was used for labeling for 1 h at room temperature, which was followed by anti-mouse Alexa488 (A21202, Life Technologies) (1∶300) labeling for 1 h at 37°C. After washings, images were taken with a Leica SP5 AOBS confocal laser scanning microscope, using the 488 nm argon laser line for excitation and 500–530 nm spectral filter for emission detection. Images were analyzed with ImageJ software (http://rsbweb.nih.gov/ij/): whole cells and plasma membranes (PMs) were drawn around for at least 15 cells/3 views for each treatment. The average intensity of the pixels representing the PM was divided by the average intensity of the whole cell in an equatorial focal plane. The Student’s t-test was performed. Experiments were repeated 3 times; the results demonstrated the same tendency. A linear region of interest was chosen for every representative image. The fluorescence intensity of the pixels within this region was plotted.

Gungor B., Gombos I., Crul T., Ayaydin F., Szabó L., Török Z., Mátés L., Vígh L, & Horváth I. (2014). Rac1 Participates in Thermally Induced Alterations of the Cytoskeleton, Cell Morphology and Lipid Rafts, and Regulates the Expression of Heat Shock Proteins in B16F10 Melanoma Cells. PLoS ONE, 9(2), e89136.

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Immunohistochemical Analysis of Neuronal Markers

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Immunohistochemistry was performed as previously described.⁴⁷ After fixation in 4% PFA at 4°C, overnight, the brain was fully preserved in 30% sucrose PBS solution for cryoprotection. Then, 40‐μm slices from the brains were cut in a frozen microtome equipment later. The slices were permeabilized with 0.1% Triton X‐100 for 15 min, then blocked in 5% bovine serum albumin (BSA) for another hour at room temperature. For immunolabeling, the slices were incubated with indicated primary antibodies: anti‐CaMKII (1:300, Fukunaga et al., 1988), anti‐phospho‐Thr286‐CaMKII (1:300, Fukunaga et al., 1988), anti‐calcineurin (1:300, Fukunaga et al., 1988), anti‐MAP2 (1:1000, Millipore, Cat# 05‐346), anti‐NEUN (1:500, Millipore, Cat# ABN78), anti‐phospho‐Ser603‐Synapsin1 (1:300, Millipore, AB5583), and anti‐PSD95 (1:300, Thermo, MA1‐045). After incubation for two nights at 4°C, slices were incubated with Alexa Fluor 488‐conjugated anti‐mouse IgG (1:300, A21202, Life Technologies) and Alexa Fluor 594‐conjugated anti‐rabbit IgG (1:300, A21207, Life Technologies) for 1 h at RT. Using Zeiss LSM 800 confocal microscope for images and fluorescence values and co‐localization were later analyzed with ImageJ software.

Li Z., Liu X., Li C., Wei Z., Shi Y., Song H., Chen X., Zhang Y., Li J., Zhu R., Hu B., Ye W., Huo D., Jiang G., Sasaki T., Zhang L., Han F, & Lu Y. (2022). Decreased synapse‐associated proteins are associated with the onset of epileptic memory impairment in endothelial CDK5‐deficient mice. MedComm, 3(3), e128.

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Cell Proliferation Quantification by BrdU Incorporation

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Cell proliferation was assessed by quantification of cells actively synthesizing DNA. HCT116 p53+/+ and HCT116 p53−/− cells treated with 10 μM Nutlin-3 as indicated. 60 min prior to harvesting cells, 1 mg/ml of BrdU was added to cultivation medium. After trypsinization, cells were fixed with 70% ethanol. DNA denaturation was achieved by incubation with 2M HCl with 0.5% Triton X-100 for 10 min at 37°C. Before staining with anti-BrdU antibody (sc-51514; Santa Cruz Biotechnology), samples were neutralized with 0.1M Na₂B₄O₇. Following incubation with secondary antibody (A21202, Life Technologies). BrdU positivity was analyzed by flow cytometry. Data represent average numbers of BrdU positive cells from three independent experiments (each run in duplicate). T test was used for statistical analysis; an asterisk denotes significance at p<0.001.

Allen M.A., Andrysik Z., Dengler V.L., Mellert H.S., Guarnieri A., Freeman J.A., Sullivan K.D., Galbraith M.D., Luo X., Kraus W.L., Dowell R.D, & Espinosa J.M. (2014). Global analysis of p53-regulated transcription identifies its direct targets and unexpected regulatory mechanisms. eLife, 3, e02200.

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Cytoskeleton Analysis in Neonatal Rat Cardiomyocytes

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NRVMs were analyzed using standard methods (Mansour et al. 2004 (link)). Immunolabelling used primary anti-PKCε antibody (1:200) (Abcam, #ab63638) or α-actinin (Abcam, #ab9465), and secondary antibody (1:500) (Life Technologies, #A21202, Donkey anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor® 488 conjugate, and #A21207, Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor® 594 conjugate). The cytoskeleton remained after removal of membranes, cytosol, and nuclei and was observed by microscopy (Li and Russell 2013 (link)).

Lin Y.H., Swanson E.R., Li J., Mkrtschjan M.A, & Russell B. (2015). Cyclic mechanical strain of myocytes modifies CapZβ1 post translationally via PKCε. Journal of muscle research and cell motility, 36(4-5), 329-337.

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Immunocytochemical Analysis of Stem Cell Markers

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Cells were fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.25% Triton X-100 and blocked with 5% FCS in PBS for 1 h. The fixed cells were incubated overnight at 4 °C in PBS + 1% FCS with antibodies mouse anti-OCT4 (1:500, ab18976, Abcam, Cambridge, MA), rabbit anti-SOX2 (1:500, MA516399, ThermoFisher), rabbit anti-TUJ1 (1:500, MAB1195, R&D system, Minneapolis, MN), rabbit anti-OLIG2 (1:500, NBP128667, Novus), rabbit anti-HB9 (1:500, ABN174, Millipore-Sigma, Temecula, CA), rabbit anti-CHAT (1:500, AB144P, Abcam, Cambridge, MA), and rabbit anti-cleaved caspase-3 (CC3, 1:500, 9669S, Cell Signaling Technology) followed by incubation with secondary antibodies: FITC-conjugated anti-mouse IgG (1:1000, A21202, Life Technologies), FITC-conjugated anti-rabbit IgG (1:1000, A11034, Life Technologies), Cy3-conjugated anti-mouse IgG (1:1000, A11003, Life Technologies), and Cy3-conjugated anti-rabbit IgG (1:1000, A11035, Life Technologies). The treated cells were covered with Slowfade antifade with DAPI (Life Technologies) for nuclear staining and covered with a glass coverslip. Images were captured with a fluorescence microscope (DM5000B, Leica, Wetzlar, Germmany).

Kim D., Kim S., Sung A., Patel N., Wong N., Conboy M.J, & Conboy I.M. (2022). Autologous treatment for ALS with implication for broad neuroprotection. Translational Neurodegeneration, 11, 16.

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Immunofluorescence Staining of Neuronal Markers

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Cells were fixed with 4% paraformaldehyde and then blocked in PBS containing 10% FBS and 0.1% Triton X-100 for 1 h at 37 °C. Primary antibodies against PSD95 (mouse monoclonal antibody, 1:100, catalog #: MAB1596, PMID: 25,498,153, Sigma-Aldrich, St.Louis, USA), Map2 (mouse monoclonal antibody, 1:500, catalog #: M9942, PMID: 26,903,822, Sigma‒Aldrich, St.Louis, USA; rabbit polyclonal antibody, 1:500, catalog #: ab32454, Abcam, Cambridge, UK) and synapsin-1 (rabbit monoclonal antibody, 1:100, catalog #: ab254349, PMID: 9,539,796, Abcam, Cambridge, UK) were incubated with the cells overnight at 4 °C. Then, appropriate fluorescent secondary antibodies (A-21,202, A-21,207 or A-21,206, Life Technologies, California, USA) diluted 1:1000 were incubated with the cells for 1 h at 37 °C in the dark. Following DAPI staining, the cells were mounted and analysed under confocal laser scanning microscopy (SP8, Leica, Wetzlar, Germany).

Huang Y.Q., Gu X., Chen X., Du Y.T., Chen B.C, & Sun F.Y. (2023). BMECs Ameliorate High Glucose-Induced Morphological Aberrations and Synaptic Dysfunction via VEGF-Mediated Modulation of Glucose Uptake in Cortical Neurons. Cellular and Molecular Neurobiology, 43(7), 3575-3592.

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Immunolabeling of Micro-Tissue Engineered Neural Networks

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Following fixation in 4% formaldehyde for 35 min, micro-TENNs were rinsed in PBS, blocked, and permeabilized with 0.3% Triton X100 plus 4% horse serum for 60 min. Primary antibody incubation was performed in PBS with 4% serum at 4 °C for 12 h. Cells were immunolabeled for: (1) MAP-2 (AB5622, 1:100, Millipore, Billerica, MA) a microtubule-associated protein expressed primarily in neuronal somata and dendrites; and (2) TUJ1/beta III Tubulin (T8578, 1:500, Sigma-Aldrich, St. Louis, MO), an element of microtubules expressed in neurites. Subsequent to rinsing, fluorescent secondary antibodies (1:500 Alexa-488, A21202, or Alexa-647, A313573, Life Technologies, Waltham, MA) were added at 18°C–24 °C for 2 h. Finally, Hoechst (1:10 000, H3570, Life Technologies, Waltham, MA) was added at 18 °C–24 °C for 10 min. Labeled micro-TENNs were fluorescently imaged using a LSM 710 confocal microscope with a Plan-Apochromat 10x/0.45 lens (Carl Zeiss, Jena, Germany). To acquire the image, 120 slices of 2.59 μm each were acquired for each channel with 8 bit resolution. The images presented are maximum projections of the series of confocal slices.

Harris J.P., Struzyna L.A., Murphy P.L., Adewole D.O., Kuo E, & Cullen D.K. (2016). Advanced biomaterial strategies to transplant preformed micro-tissue engineered neural networks into the brain. Journal of neural engineering, 13(1), 016019.

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