A 21235

Manufactured by Thermo Fisher Scientific

Sourced in United States

The A-21235 is a laboratory instrument designed for performing high-precision analytical measurements. It is a specialized piece of equipment used in scientific research and industrial applications that require accurate and reliable data collection.

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Goat anti-mouse Alexa-Fluor 647 (A-21235; (2 citations)

69 protocols using a 21235

Quantifying α-Synuclein Expression by Flow Cytometry

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Flow cytofluorometry was used to correlate αsyn expression to the fluorescence resulting from the Venus YFP protein-fragment complementation. After 48h incubation with or without 1μg/ml tetracycline, cells were suspended in PBS supplemented with 1% bovine serum albumin and 2.5% goat serum and subsequently fixed and permeabilized following supplier's instructions (Leucoperm, AbD serotec, USA). Cells were incubated for 1h at RT with anti-αsyn antibodies (1:500; syn-1, 610787, BD) followed by 1h at RT with Alexa Fluor®647-conjugated anti-mouse IgG secondary antibodies (1:1000; A21235, Life tech). Unstained cells and control stainings with isotype control antibodies were also performed. The samples were measured on a flow cytometer (Accuri™C6, BD) and data analysis was performed using FlowJo software (Tree Star Inc., USA).

Moussaud S., Malany S., Mehta A., Vasile S., Smith L.H, & McLean P.J. (2015). Targeting alpha-synuclein oligomers by protein-fragment complementation for drug discovery in synucleinopathies. Expert opinion on therapeutic targets, 19(5), 589-603.

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Immunohistochemistry of Brain Tissues

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Mice were anesthetized with Avertin (250 mg/kg body weight) and transcardially perfused with 4% formaldehyde (EMS 15713) in 1x PBS, pH 7.6. The brains were dissected, post-fixed for 2 hours at room temperature and processed through 10% sucrose in PBS overnight followed by 30% sucrose in PBS until they sink. These cryoprotected brains were embedded in 2% gelatin (Sigma G2500), 0.9% NaCl in a metal mold, quickly frozen on a metal block cooled with liquid nitrogen, sectioned with Leica CM1900 at 20 μm thickness and mounted on SuperFrost Gold Plus microscopy slides (Fisher Scientific). The sections were incubated with primary antibodies, chicken anti-GFP (Aves GFP-1020), rabbit anti-RFP (Rockland 600-401-379) and mouse anti-Cre Recombinase, Clone 2D8 (Millipore MAB3120), diluted in Cyto-Q Immuno Diluent and Block (Innovex Biosciences). The secondary antibodies were anti-chicken AF488, anti-rabbit AF555 and anti-mouse AF647, all made in goat (Life Technologies A11039, A21428 and A21235, respectively). The slides were stained with DAPI, mounted with Fluoromount-G (Southern Biotech) and image acquisition was performed with Leica TCS SP5 confocal microscope. The images were analyzed processed with Imaris 7.6.5 (Bitplane).

Gee J.M., Smith N.A., Fernandez F.R., Economo M.N., Brunert D., Rothermel M., Morris S.C., Talbot A., Palumbos S., Ichida J.M., Shepherd J.D., West P.J., Wachowiak M., Capecchi M.R., Wilcox K.S., White J.A, & Tvrdik P. (2014). Imaging Activity in Neurons and Glia with a Polr2a-based and Cre-dependent GCaMP5G-IRES-tdTomato Reporter Mouse. Neuron, 83(5), 1058-1072.

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Immunofluorescence Analysis of Inguinal WAT

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Inguinal white adipose tissue (iWAT) was fixed in 10% phosphate-buffered formalin and embedded in paraffin. Immunofluorescence was carried out using rabbit anti-mouse synaptophysin (1:300) (17785-1-AP, Proteintech) and mouse anti-mouse tyrosine hydroxylase (1:200) (MAB318, EMD Millipore) primary antibodies. Primary antibodies were incubated overnight at 4°C. The secondary antibodies used were AlexaFluor 488 donkey anti-rabbit and 594 goat anti-rabbit (A11037, A11072, A11020, A21235, Life Technologies) at a 1:400 dilution, and were incubated for 1 h at room temperature. Images were either captured with an inverted fluorescence microscope (IX51 Olympus) or with a confocal laser scanning microscope (Zeiss LSM 710). Hematoxylin and eosin (H&E) images were acquired with the EVOS M7000 Imaging system (Thermo Fisher System). Synaptophysin, tyrosine hydroxylase and griffonia simplicifolia agglutinin-I were quantified using CellProfiler 3.0 (http://cellprofiler.org). Innervation and vascularization densities were quantified as the number of sprinkles normalized to adipocytes count (mm²/adipocytes).

Nigro P., Vamvini M., Yang J., Caputo T., Ho L.L., Carbone N.P., Papadopoulos D., Conlin R., He J., Hirshman M.F., White J.D., Robidoux J., Hickner R.C., Nielsen S., Pedersen B.K., Kellis M., Middelbeek R.J, & Goodyear L.J. (2023). Exercise training remodels inguinal white adipose tissue through adaptations in innervation, vascularization, and the extracellular matrix. Cell reports, 42(4), 112392.

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Quantitative Immunofluorescence Analysis of Kidney Proteins

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Immunofluorescence analyses were performed as previously described 3. Briefly, the kidneys were obtained from C57BL6 mice at indicated ages, fixed in 4% paraformaldehyde in PBS for 4 hrs at 4°C, subjected to 30% sucrose overnight at 4°C, embedded in OCT compound (Tissue‐Tek, Miles) and sectioned at 6 μm (Leica CM1850; Leica Instruments, Wetzlar, Germany). Slides were washed 3 × 5 min. in PBS and blocked with 10% serum of the secondary antibody host species for 1 hr. And then slides were incubated with primary antibodies at 4°C overnight as follows: Notch1, Beclin‐1, LC3 and nephrin. Dilution and other supplementary information of the primary and secondary antibodies are as follows.

Dilution of primary antibodies:

Notch1 (ab52627; Abcam): 1:500;

LC3 (M186‐3; MBL): 1:500

P62 (ab56416): 1:500

The secondary antibodies:

The Alexa 488‐conjugated goat antimouse IgG secondary antibody (CA11008S; Invitrogen) and 647‐conjugated goat anti‐rabbit IgG secondary antibody (A21235; Life Technologies) as well as the Alexa 594‐conjugated goat antimouse IgG secondary antibody (CA11008S) were used at 1:1000.

Imaging was conducted with a confocal microscope (Olympus FV1000; Tokyo, Japan), and the fluorescent intensity was quantified using IMAGEJ software (National Institutes of Health, Bethesda, MD, USA).

Zhang C., Li W., Wen J, & Yang Z. (2017). Autophagy is involved in mouse kidney development and podocyte differentiation regulated by Notch signalling. Journal of Cellular and Molecular Medicine, 21(7), 1315-1328.

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Immunofluorescence Staining Protocols for SARS-CoV-2 Research

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The mouse and rabbit polyclonal Ab against SARS-CoV-2 N protein was homemade and used at a dilution of 1:500 when labeling. Purchased antibody data are shown in Table 1.Table 1

Source of antibodies and dyes with work concentration for immunofluorescence.

Antibodies and dyes	Source and work concentration
ACE2	Abcam (ab15348), rabbit polyclonal, 5 µg/ml
ACE2	Bioss (bs-1004R), rabbit polyclonal (1:100)
ACE2	Sino biologicals (10108-T56), rabbit polyclona (1:100)
Tubulin-IV	Abcam (ab179504), rabbit monoclonal (1:500)
Tubulin-IV	Abcam (ab11315), mouse monoclonal, 5 µg/ml
Muc5AC	Abcam (ab178294), rabbit monoclonal (1:250)
Muc5AC	Bioss (bs-7166R), rabbit polyclonal (1:250)
CCSP	Abcam (ab40873), rabbit polyclonal (1:500)
ZO-1 TJP1	Invitrogen, (402200), rabbit polyclonal, 2.5 µg/ml
Alexa Fluor^® 488, 594, 647 goat anti-mouse IgG (H + L)	Life Technologies A-10680 (1:1000); A-11005 (1:1000); A-21235 (1:1000)
Alexa Fluor^® 488, 594, 647 goat anti-rabbit IgG (H + L)	Life Technologies A-11008 (1:1000); A-11012 (1:1000); A-21244 (1:1000)
DAPI	Abcam (ab228594), 1:1000

Zhu N., Wang W., Liu Z., Liang C., Wang W., Ye F., Huang B., Zhao L., Wang H., Zhou W., Deng Y., Mao L., Su C., Qiang G., Jiang T., Zhao J., Wu G., Song J, & Tan W. (2020). Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells. Nature Communications, 11, 3910.

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Immunocytochemistry and Western Blot Antibodies

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Primary antibodies used for immunocytochemistry and/or western blots included: Homer1 (1:750, Synaptic Systems; 160003), Shank2 (1:1,000, Synaptic Systems; 162204), Shank3 (1:500, Synaptic Systems; 162302 and 162304), GluA1 and GluA2 [1:8 for surface staining and 1:100 for whole cell staining, Millipore; PC246 and MAB397], VGluT1 (1:100, NeuroMab; N28/9), Shank2 (1:100, Neuromab; N23B/6), Shank3 (1:100, NeuroMab; N367/62), PSD-95 (1:100, NeuroMab; K28/43), MAP2 (1:5,000, Abcam; ab5392), green fluorescent protein (1:1,000, Abcam; ab13970), actin (1:1,000, Abcam; ab8227), and Shank2 (1:250, Cell Signaling; 12218). A custom-made VGluT1 antibody (1:500, polyclonal rabbit) was generously provided by Dr. Richard Reimer (Stanford University). All secondary antibodies (1:500, A11029, A11034, A11036, A11039, A11041, A11075, A21235 and A21449) were obtained from Life Technologies with the exception of the Dylight-350 antibody (1:250, Thermo Fisher; SA5-10069) and the HRP-conjugated antibodies (1:10,000, rabbit, mouse or guinea pig; Jackson ImmunoResearch; 706-035-148, 115-035-003 and 111-035-144).

Ha H.T., Leal-Ortiz S., Lalwani K., Kiyonaka S., Hamachi I., Mysore S.P., Montgomery J.M., Garner C.C., Huguenard J.R, & Kim S.A. (2018). Shank and Zinc Mediate an AMPA Receptor Subunit Switch in Developing Neurons. Frontiers in Molecular Neuroscience, 11, 405.

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Mitochondria-ER Colocalization Analysis

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For colocalization analysis of mitochondria and ER, native fibroblasts were fixed with 4% paraformaldehyde for 15 min and then labeled with antibodies against Tom20 (sc-17764, dilution 1:500, secondary antibody:goat anti-mouse Alexa Fluor 647; Santa Cruz Biotechnologies; A-21235, dilution 1:1000; Life Technologies) and PDI (2446S, dilution 1:1000, secondary antibody:goat anti-rabbit Alexa Fluor 488; Cell Signaling Technology; A-1000, dilution 1:1000; Life Technologies).

Grossmann D., Berenguer-Escuder C., Bellet M.E., Scheibner D., Bohler J., Massart F., Rapaport D., Skupin A., Fouquier d'Hérouël A., Sharma M., Ghelfi J., Raković A., Lichtner P., Antony P., Glaab E., May P., Dimmer K.S., Fitzgerald J.C., Grünewald A, & Krüger R. (2019). Mutations in RHOT1 Disrupt Endoplasmic Reticulum–Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease. Antioxidants & Redox Signaling, 31(16), 1213-1234.

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Quantifying Extracellular Matrix Deposition

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Monolayers were fixed with 4% formaldehyde for 10 min at RT, then blocked with 3% BSA in PBS for 1 h. Immunofluorescence was carried out using the following primary antibodies: rabbit anti-human Col IV (1:500), mouse anti-human heparan sulfate proteoglycan 2 [A76] (HSPG) (1:200), mouse anti-human paxillin (1:200) (6586, 26265, 3127, Abcam). Primary antibodies were incubated overnight at 4 °C. Secondary antibodies used were AlexaFluor® 488 chicken anti-rabbit, 594 goat anti-rabbit, 594 goat anti-mouse and 647 goat anti-mouse (A21441, A11072, A11020, A21235, Life Technologies) at 1:400 dilution, incubation of 1 h at RT. Cell nuclei were counterstained with 0.5 μg/ml DAPI and cytoplasmic lipids with 0.1 μg/ml BODIPY 493/503 (D3922, Life Technologies). Images were either captured with an IX71 inverted fluorescence microscope (Olympus) or with a confocal laser scanning microscope (LSM510, Zeiss, Germany). Deposition of Col IV and HSPG were quantified by adherent cytometry and as previously described27 (link). Extent of ECM deposition was quantified by area of fluorescence normalized to nuclei count (μm²/nuclei).

Lee M.H., Goralczyk A.G., Kriszt R., Ang X.M., Badowski C., Li Y., Summers S.A., Toh S.A., Yassin M.S., Shabbir A., Sheppard A, & Raghunath M. (2016). ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs. Scientific Reports, 6, 21173.

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Immunostaining of Nuclear Pore Proteins

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For all immunostainings, primary antibodies (Abcam) used were rabbit anti-Nup358 at 1:200 (ab64276); rabbit anti-Nup214 at 1:200 (ab70497); mouse anti-Nup62 at 1:300 (ab96134); rabbit anti-Nup153 at 1:300 (ab171074); rabbit anti-TPR at 1:100 (ab170940); rabbit anti-lamin A at 1:200 (ab26300). Secondary antibodies: the secondary antibodies used were anti-mouse Cy3 (ab97035), anti-rabbit Cy3 (ab6939; Abcam), Alexa Fluor 647 goat anti-mouse (A21235), and Alexa Fluor 594 goat anti-rabbit (A11072; Molecular Probes).

Ashkenazy-Titelman A., Atrash M.K., Boocholez A., Kinor N, & Shav-Tal Y. (2022). RNA export through the nuclear pore complex is directional. Nature Communications, 13, 5881.

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Immunohistochemistry of Neuronal Markers

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Primary antibodies used were as follows: biotinylated goat anti-GFP (1:400; Abcam, catalog #ab6658), rabbit anti-TBR1 (1:200; Abcam, catalog #ab31940), rat anti-CTIP2 (1:200; Abcam, catalog #ab18465), and mouse anti-SATB2 (1:200; Abcam, catalog #ab51502). Secondary antibodies used were as follows: streptavidin Alexa-488 (1:800; Invitrogen, catalog #S32354) for GFP. Goat anti-rabbit antibody conjugated to Alexa-488 (1:400, Molecular Probes, catalog #A11008) for TBR1. Goat anti-rat antibody conjugated to Alexa-568 (1:400, Molecular Probes, catalog #A11077) for CTIP2. Goat anti-mouse antibody conjugated to Alexa-647 (1:400, Molecular Probes, catalog #A21235) for SATB2. Tissue processing for immunohistochemistry was performed as described by Subramanian et al. (2011 (link)). For each control and experimental condition, ≥100 cells were counted from each of five biological replicates for Figure 1 and from each of three biological replicates for Figure 5.

Muralidharan B., Khatri Z., Maheshwari U., Gupta R., Roy B., Pradhan S.J., Karmodiya K., Padmanabhan H., Shetty A.S., Balaji C., Kolthur-Seetharam U., Macklis J.D., Galande S, & Tole S. (2017). LHX2 Interacts with the NuRD Complex and Regulates Cortical Neuron Subtype Determinants Fezf2 and Sox11. The Journal of Neuroscience, 37(1), 194-203.

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