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Alexa 488 labeled secondary antibody

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The Alexa 488-labeled secondary antibody is a fluorescently-labeled antibody that is designed to bind to primary antibodies and emit green fluorescence when excited by a light source. This secondary antibody is commonly used in immunoassays, such as immunofluorescence microscopy, to detect and visualize the presence and location of target proteins or other biomolecules in cells or tissue samples.

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Lab products found in correlation

Axiovert 200m, by Zeiss (4 mentions) Dapi mounting media, by Vector Laboratories (2 mentions) Primary anti 53bp1 antibody, by Santa Cruz Biotechnology (2 mentions) Pna cy3 telo c probes, by Agilent Technologies (2 mentions) Rabbit anti γh2ax antibody, by Santa Cruz Biotechnology (2 mentions) Facscalibur, by BD (2 mentions) Lsm 880 confocal microscope, by Zeiss (2 mentions) Axio observer d1 inverted fluorescence microscope, by Zeiss (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) γh2ax antibody, by Merck Group (1 mentions) Winlist software, by Verity Software House (1 mentions) Hif 1α, by Abcam (1 mentions) Ts100 f fluorescence microscope, by Nikon (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions) Nis elements microscope imaging software, by Nikon (1 mentions) Facscanto 2 platform, by BD (1 mentions) Ebioscience foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions) Vicell xr automatic cell counter, by Beckman Coulter (1 mentions) 780 confocal microscope, by Zeiss (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions)

22 protocols using alexa 488 labeled secondary antibody

1

Immunofluorescence Analysis of DNA Damage Markers

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Primary fibroblasts from healthy controls, CTC1 mutation positive patients and LCC patients were fixed for 5 minutes in 4% vol/vol formaldehyde in H2O and permeabilized in PBS with 1% BSA 0.1% Triton X100. Cells were incubated with primary anti-53BP1 antibody (SantaCruz) for 1 hour at room temperature, and then with Alexa 488 labeled secondary antibody (Life Technology). Samples were fixed for 5 minutes in 4% vol/vol paraformaldehyde and dehydrated in successive 5 minutes baths of 70% Ethanol, 95% ethanol and 100% ethanol. PNA-cy3-Telo-C probes (DAKO) were hybridized according to the supplier’s recommendations. Briefly, probes were incubated with the samples for 5 minutes at 80°C, and left in the dark at room temperature for 90 minutes. Samples were then washed twice in 70% formamide, 10 mM Tris-HCL, and PBS, and mounted with DAPI mounting media (Vectashield).
Jenkinson E.M., Rodero M.P., Kasher P.R., Uggenti C., Oojageer A., Goosey L.C., Rose Y., Kershaw C.J., Urquhart J.E., Williams S.G., Bhaskar S.S., O’Sullivan J., Baerlocher G.M., Haubitz M., Aubert G., Barañano K.W., Barnicoat A.J., Battini R., Berger A., Blair E.M., Brunstrom-Hernandez J.E., Buckard J.A., Cassiman D.M., Caumes R., Cordelli D.M., De Waele L.M., Fay A.J., Ferreira P., Fletcher N.A., Fryer A.E., Goel H., Hemingway C.A., Henneke M., Hughes I., Jefferson R.J., Kumar R., Lagae L., Landrieu P.G., Lourenço C.M., Malpas T.J., Mehta S.G., Metz I., Naidu S., Õunap K., Panzer A., Prabhakar P., Quaghebeur G., Schiffmann R., Sherr E.H., Sinnathuray K.R., Soh C., Stewart H.S., Stone J., Van Esch H., Van Mol C.E., Vanderver A., Wakeling E.L., Whitney A., Pavitt G.D., Griffiths-Jones S., Rice G.I., Revy P., van der Knaap M.S., Livingston J.H., O’Keefe R.T, & Crow Y.J. (2016). Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts. Nature genetics, 48(10), 1185-1192.
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2

Immunofluorescence Analysis of DNA Damage Markers

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Primary fibroblasts from healthy controls, CTC1 mutation positive patients and LCC patients were fixed for 5 minutes in 4% vol/vol formaldehyde in H2O and permeabilized in PBS with 1% BSA 0.1% Triton X100. Cells were incubated with primary anti-53BP1 antibody (SantaCruz) for 1 hour at room temperature, and then with Alexa 488 labeled secondary antibody (Life Technology). Samples were fixed for 5 minutes in 4% vol/vol paraformaldehyde and dehydrated in successive 5 minutes baths of 70% Ethanol, 95% ethanol and 100% ethanol. PNA-cy3-Telo-C probes (DAKO) were hybridized according to the supplier’s recommendations. Briefly, probes were incubated with the samples for 5 minutes at 80°C, and left in the dark at room temperature for 90 minutes. Samples were then washed twice in 70% formamide, 10 mM Tris-HCL, and PBS, and mounted with DAPI mounting media (Vectashield).
Jenkinson E.M., Rodero M.P., Kasher P.R., Uggenti C., Oojageer A., Goosey L.C., Rose Y., Kershaw C.J., Urquhart J.E., Williams S.G., Bhaskar S.S., O’Sullivan J., Baerlocher G.M., Haubitz M., Aubert G., Barañano K.W., Barnicoat A.J., Battini R., Berger A., Blair E.M., Brunstrom-Hernandez J.E., Buckard J.A., Cassiman D.M., Caumes R., Cordelli D.M., De Waele L.M., Fay A.J., Ferreira P., Fletcher N.A., Fryer A.E., Goel H., Hemingway C.A., Henneke M., Hughes I., Jefferson R.J., Kumar R., Lagae L., Landrieu P.G., Lourenço C.M., Malpas T.J., Mehta S.G., Metz I., Naidu S., Õunap K., Panzer A., Prabhakar P., Quaghebeur G., Schiffmann R., Sherr E.H., Sinnathuray K.R., Soh C., Stewart H.S., Stone J., Van Esch H., Van Mol C.E., Vanderver A., Wakeling E.L., Whitney A., Pavitt G.D., Griffiths-Jones S., Rice G.I., Revy P., van der Knaap M.S., Livingston J.H., O’Keefe R.T, & Crow Y.J. (2016). Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts. Nature genetics, 48(10), 1185-1192.
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3

Neutrophil Activation and Imaging

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Neutrophils were incubated with either propofol or lipid emulsion for 30 min at 37°C with 5% CO2 prior to addition of 25 nM phorbol 12-myristate 13-acetate (PMA; Sigma). Following an additional 2-h incubation at 37°C with 5% CO2, cells were fixed via addition of paraformaldehyde (4% final concentration). Fixed neutrophils were blocked with PBS plus 2% bovine serum albumin and goat serum, followed by staining using an anti-human myeloperoxidase antibody (1:300; Dako Denmark) and an Alexa 488-labeled secondary antibody (1:500; Life Technologies) as previously described (Corriden et al., 2015 (link)). Cells were imaged using an AxioObserver D1 Inverted Fluorescence Microscope (Zeiss) equipped with an LD A-Plan 20X/0.35 Ph1 objective and processed with affinity photo (Serif).
Meier A., Chien J., Hobohm L., Patras K.A., Nizet V, & Corriden R. (2019). Inhibition of Human Neutrophil Extracellular Trap (NET) Production by Propofol and Lipid Emulsion. Frontiers in Pharmacology, 10, 323.
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4

Cell Cycle Analysis of DNA Damage Response

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HCT116 and HT-29 cells (1×106/plate) were seeded on 100 mm dishes and cultured at 37°C overnight. The next day, cells were incubated with either MLN0905 or DMSO for the indicated times. Adherent and floating cells were harvested and fixed in 70% ethanol and stored overnight at -20°C. Cells were centrifuged and cell pellets washed with 0.5% BSA in PBS and then stained with γH2AX antibody (Millipore Corporation, Billerica, MA) per the manufacturer's instructions and incubated overnight at 4°C. Pellets were then washed and stained with the Alexa-488 labeled secondary antibody (Molecular Probes, Life Technologies Corporation, Bedford, MA) diluted 1∶150 and incubated for 30 minutes at room temperature. Pellets were washed and re-suspended in propidium iodide and RNaseA in PBS. Cell-cycle distributions were determined using flow cytometry (FACS Calibur, Becton Dickinson, Franklin Lakes, NJ) and analyzed using Winlist software (Verity Software House, Topsham, ME).
Driscoll D.L., Chakravarty A., Bowman D., Shinde V., Lasky K., Shi J., Vos T., Stringer B., Amidon B., D'Amore N, & Hyer M.L. (2014). Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines. PLoS ONE, 9(11), e111060.
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5

γH2AX and Telomere Analysis in EBV Cells

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EBV immortalized cells derived from cryopreserved lymphocytes were spun onto Cytospin microscopic slides at 200 rpm for 3 minutes. Cells were fixed in 2% paraformaldehyde, permeabilized with 0.5% triton X-100, and blocked with 10% FBS for 1-2 hours. Cells were stained with a rabbit anti-γH2AX antibody (1:200, Santa Cruz) 51 (link), followed by Alexa 488-labeled secondary antibody (1:500; Molecular Probes). For combined telomere FISH, slides were fixed in 2% paraformaldehyde and then used to perform telomere-FISH. Z-stack images were captured on a fluorescence microscope (Axiovert 200M; Carl Zeiss). The experiments were repeated three times on each sample.
Shi J., Yang X.R., Ballew B., Rotunno M., Calista D., Fargnoli M.C., Ghiorzo P., Paillerets B.B., Nagore E., Avril M.F., Caporaso N.E., McMaster M.L., Cullen M., Wang Z., Zhang X., Bruno W., Pastorino L., Queirolo P., Banuls-Roca J., Garcia-Casado Z., Vaysse A., Mohamdi H., Riazalhosseini Y., Foglio M., Jouenne F., Hua X., Hyland P.L., Yin J., Vallabhaneni H., Chai W., Minghetti P., Pellegrini C., Ravichandran S., Eggermont A., Lathrop M., Peris K., Scarra G.B., Landi G., Savage S.A., Sampson J.N., He J., Yeager M., Goldin L.R., Demenais F., Chanock S.J., Tucker M.A., Goldstein A.M., Liu Y, & Landi M.T. (2014). Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nature genetics, 46(5), 482-486.
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6

HIF-1α Immunofluorescence Staining

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Cells cultured under normoxic and hypoxic conditions were washed in PBS and fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min. The cells were permeabilized with 0.05% Triton X-100 solution at room temperature for 10 min and blocked with 5% normal goat serum (NGS) at room temperature for 1 h. Then, the cells were stained with specific primary antibodies against HIF-1α (Abcam, Cambridge, UK) for >12 h and incubated for 2 h with Alexa 488-labeled secondary antibody (1:1000; Molecular Probes, Eugene, OR, USA). The counterstaining of nuclei was conducted with DAPI.
Kang I., Lee B.C., Choi S.W., Lee J.Y., Kim J.J., Kim B.E., Kim D.H., Lee S.E., Shin N., Seo Y., Kim H.S., Kim D.I, & Kang K.S. (2018). Donor-dependent variation of human umbilical cord blood mesenchymal stem cells in response to hypoxic preconditioning and amelioration of limb ischemia. Experimental & Molecular Medicine, 50(4), 35.
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7

Immunofluorescent Staining of OPC Cultures

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OPC cultures were fixed with 4% paraformaldehyde. Fixed cells were incubated in primary antibody overnight at 4°C, washed thoroughly and incubated with Alexa 488-labeled secondary antibody (1:800; Molecular Probes) for 2 hours at room temperature.
Chen Y., Zhen W., Guo T., Zhao Y., Liu A., Rubio J.P., Krull D., Richardson J.C., Lu H, & Wang R. (2017). Histamine Receptor 3 negatively regulates oligodendrocyte differentiation and remyelination. PLoS ONE, 12(12), e0189380.
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8

γH2AX and Telomere Analysis in EBV Cells

Cited 1 time
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EBV immortalized cells derived from cryopreserved lymphocytes were spun onto Cytospin microscopic slides at 200 rpm for 3 minutes. Cells were fixed in 2% paraformaldehyde, permeabilized with 0.5% triton X-100, and blocked with 10% FBS for 1-2 hours. Cells were stained with a rabbit anti-γH2AX antibody (1:200, Santa Cruz) 51 (link), followed by Alexa 488-labeled secondary antibody (1:500; Molecular Probes). For combined telomere FISH, slides were fixed in 2% paraformaldehyde and then used to perform telomere-FISH. Z-stack images were captured on a fluorescence microscope (Axiovert 200M; Carl Zeiss). The experiments were repeated three times on each sample.
Shi J., Yang X.R., Ballew B., Rotunno M., Calista D., Fargnoli M.C., Ghiorzo P., Paillerets B.B., Nagore E., Avril M.F., Caporaso N.E., McMaster M.L., Cullen M., Wang Z., Zhang X., Bruno W., Pastorino L., Queirolo P., Banuls-Roca J., Garcia-Casado Z., Vaysse A., Mohamdi H., Riazalhosseini Y., Foglio M., Jouenne F., Hua X., Hyland P.L., Yin J., Vallabhaneni H., Chai W., Minghetti P., Pellegrini C., Ravichandran S., Eggermont A., Lathrop M., Peris K., Scarra G.B., Landi G., Savage S.A., Sampson J.N., He J., Yeager M., Goldin L.R., Demenais F., Chanock S.J., Tucker M.A., Goldstein A.M., Liu Y, & Landi M.T. (2014). Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma. Nature genetics, 46(5), 482-486.
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9

Caspase-3 Activation Detection Methods

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Cleaved‐activated caspase‐3 was detected in cell transfectants grown for 48 h in suspension by western blot analysis using a specific mAb (Cell Signaling Technology, Frankfurt, Germany), according to the manufacturer`s protocol and by immunofluorescent staining upon use of an Alexa 488‐labeled secondary antibody (Thermo Fisher Scientific, Carlsbad, CA, USA). In addition, caspase activation was detected indirectly, by measuring its cleavage products of cytokeratin‐18 utilizing the M30 CytoDeath FACS kit (Roche Diagnostics GmbH) and by CLSM. Moreover, activated caspase was detected by binding of an irreversible fluorescence‐labeled pan‐caspase inhibitor applying the CaspACE™ FITC‐VAD‐FMK in Situ Marker kit (Promega, Madison, WI, USA).
Dolinschek R., Hingerl J., Benge A., Zafiu C., Schüren E., Ehmoser E., Lössner D, & Reuning U. (2020). Constitutive activation of integrin αvβ3 contributes to anoikis resistance of ovarian cancer cells. Molecular Oncology, 15(2), 503-522.
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10

Immunofluorescence Staining of HCV Core Protein

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Cells were fixed with a 1:1 mixture of methanol and acetone for 10 min at −20 °C, washed with 1X PBS, and blocked with 1% BSA at room temperature for 1 h. Subsequently, cells were incubated with primary antibody overnight at 4 °C, followed by secondary antibody at room temperature for 1 h. After staining of cells with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA), fluorescence was examined under a Nikon TS100-F fluorescence microscope (Tokyo, Japan) equipped with a digital camera. Fluorescence images were analyzed using Nikon NIS-Elements microscope imaging software. An antibody against HCV core protein was purchased from Anogen (Mississauga, ON, Canada). Alexa 488-labeled secondary antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Park S., Kim J.Y., Kwon H.C., Jang D.S, & Song Y.J. (2022). Antiviral Activities of Ethyl Pheophorbides a and b Isolated from Aster pseudoglehnii against Influenza Viruses. Molecules, 28(1), 41.
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