Alexa fluor 488 conjugated goat anti mouse secondary antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany

The Alexa Fluor 488-conjugated goat anti-mouse secondary antibody is a fluorescent-labeled secondary antibody used to detect and visualize primary mouse antibodies in various immunoassays and imaging applications.

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Lab products found in correlation

4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (8 mentions) Alexa fluor 594 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (6 mentions) Gtx635679, by GeneTex (5 mentions) Cytation 5, by Agilent Technologies (5 mentions) 96 well plate, by Corning (4 mentions) Alexa fluor 568 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (3 mentions) Lsm 780 confocal microscope, by Zeiss (3 mentions) Facsaria iiiu cell sorter, by BD (2 mentions) Lsrfortessa flow cytometer, by BD (2 mentions) Lsm 880 confocal microscope, by Zeiss (2 mentions) Multiphoton confocal laser scanning microscope, by Zeiss (2 mentions) Lsm 700 confocal microscope, by Zeiss (2 mentions) A11001, by Thermo Fisher Scientific (2 mentions) Jbw301 mouse monoclonal anti γh2ax antibody, by Merck Group (2 mentions) Alexa fluor 488 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions) Lsm 780, by Zeiss (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Facscalibur flow cytometer, by BD (2 mentions) Lsm 710, by Zeiss (2 mentions) Leica dmlb fluorescence microscope, by Leica Microsystems (2 mentions)

74 protocols using alexa fluor 488 conjugated goat anti mouse secondary antibody

Genetic Instability Analysis in CD34+ Cells

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Genetic instability was analyzed in CD34+ cells by immunofluorescence staining of DNA double-strand-breaks (DSB), immunofluorescence staining of centrosomes and cytogenetic analysis in CD34+ cells at day 6.
Immunofluorescence staining of DSB was performed in 1 × 10⁵ CD34+ cells using a JBW301 mouse monoclonal anti-γH2AX antibody (1:500) (#05-636, Merck KGaA) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500) (#A11001, Thermo Fisher Scientific). At least 50 nuclei were analyzed in each sample.
Immunofluorescence staining of centrosomes and mitotic spindles was performed in 1 × 10⁵ CD34+ cells using a polyclonal anti-pericentrin (1:1000) (#ab4448, Abcam, Cambridge, UK) and a monoclonal anti-α-tubulin (1:500) (#T6074, Sigma-Aldrich, St. Louis, MO, USA) antibody as well as an Alexa Fluor 555-conjugated donkey anti-rabbit (1:1000) (#A31572, Thermo Fisher scientific) and an Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500) (#A11001, Thermo Fisher Scientific). At least 50 nuclei were analyzed in each sample. Centrosomes displaying > 2 centrioles were classified as numerical aberrant and centrosomes with irregular form as structural aberrant.
Cytogenetic analysis of G-banded chromosomes was performed in CD34+ cells according to standard procedures [46 ]. At least 25 metaphases were analyzed in each sample according to ISCN 2020 [47 ].

Fabarius A., Samra V., Drews O., Mörz H., Bierbaum M., Darwich A., Weiss C., Brendel S., Kleiner H., Seifarth W., Greffrath W., Hofmann W.K., Schmitt C.A, & Popp H.D. (2022). Evidence for Recombinant GRP78, CALR, PDIA3 and GPI as Mediators of Genetic Instability in Human CD34+ Cells. Cancers, 14(12), 2883.

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Immunofluorescent Imaging of IFITM Variants

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For imaging experiments, 10⁵ HEK293T cells or IFITMdel MEFs were grown on sterilized glass coverslips in 12-well plates and transfected with 500 ng of plasmid encoding Ptm HA-IFITM variant or an empty vector control using Fugene 6 transfection reagent (Roche) following manufacturer's protocol. 24 hours post-transfection, cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 20 minutes, permeabilized with 0.1% Triton X100 in PBS for 10 minutes, and blocked with 2% FBS in PBS for 10 minutes. Cells were stained with the anti-HA primary antibody (1:1000, HA.11 Covance) and Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:1000, Life Technologies). Coverslips were mounted with Prolong Gold Antifade reagent containing DAPI (Life Technologies) and images were taken using an Olympus Fluoview FV10i confocal microscope.

Sharma A., McLaughlin RN J.r., Basom R.S., Kikawa C., OhAinle M., Yount J.S., Emerman M, & Overbaugh J. (2019). Macaque interferon-induced transmembrane proteins limit replication of SHIV strains in an Envelope-dependent manner. PLoS Pathogens, 15(7), e1007925.

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SARS-CoV-2 Immunofluorescence Imaging Protocol

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Cells infected with SARS-CoV-2 were fixed and permeabilised in 8% formaldehyde/1% Triton X-100 at designated time points postinfection. DAPI (Thermo Fisher Scientific, cat# P36395) was included in the mounting medium, and slides were stained with primary sheep anti-coronavirus antibodies (1:500) and a rabbit anti-sheep secondary antibody (Abcam cat# ab150182) at a 1:1000 dilution before imaging (Zeiss LSM 880 confocal microscope). Alternatively, fixed and permeabilised cells were labelled using a mouse anti-N (nucleocapsid) IgG antibody (SinoBiologicals; cat# 40143-MM08), which was detected using an AlexaFluor 488-conjugated goat anti-mouse secondary antibody (Life Technologies; cat# A28175). Cells were subsequently counterstained with DAPI (1 μg/ml) and mounted using ProLong Gold antifade (Life Technologies; cat# P36930). Sequential channels were acquired as z-stacks (1.2 μm step) using an Olympus FV3000 confocal microscope and processed using Imaris software (v 9.0.2; Bitplane).
For the enumeration of infected foci, the sheep anti-SARS-CoV-2 N antibody was used (at a 1:500 dilution) and visualised using donkey anti-sheep antibody (A11015, Thermo Fisher Scientific, 1:1000 dilution) prior to scanning (Celigo, Nexcelom).

Rihn S.J., Merits A., Bakshi S., Turnbull M.L., Wickenhagen A., Alexander A.J., Baillie C., Brennan B., Brown F., Brunker K., Bryden S.R., Burness K.A., Carmichael S., Cole S.J., Cowton V.M., Davies P., Davis C., De Lorenzo G., Donald C.L., Dorward M., Dunlop J.I., Elliott M., Fares M., da Silva Filipe A., Freitas J.R., Furnon W., Gestuveo R.J., Geyer A., Giesel D., Goldfarb D.M., Goodman N., Gunson R., Hastie C.J., Herder V., Hughes J., Johnson C., Johnson N., Kohl A., Kerr K., Leech H., Lello L.S., Li K., Lieber G., Liu X., Lingala R., Loney C., Mair D., McElwee M.J., McFarlane S., Nichols J., Nomikou K., Orr A., Orton R.J., Palmarini M., Parr Y.A., Pinto R.M., Raggett S., Reid E., Robertson D.L., Royle J., Cameron-Ruiz N., Shepherd J.G., Smollett K., Stewart D.G., Stewart M., Sugrue E., Szemiel A.M., Taggart A., Thomson E.C., Tong L., Torrie L.S., Toth R., Varjak M., Wang S., Wilkinson S.G., Wyatt P.G., Zusinaite E., Alessi D.R., Patel A.H., Zaid A., Wilson S.J, & Mahalingam S. (2021). A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research. PLoS Biology, 19(2), e3001091.

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Induction and Detection of KSHV Reactivation

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BC3 cells were mock-treated or treated with a final concentration of 20 ng/mL tetradecanoyl phorbol acetate (TPA, Millipore Sigma) for 36 h to induce KSHV reactivation. Cells were pelleted by centrifugation at 300 × g for 3 min and fixed with 4% PFA in PBS for 15 min at room temperature, followed by permeabilization with 0.1% (v/v) Triton X-100 in PBS with 1% (w/v) BSA for 15 min at room temperature. After washing and blocking the cells in PBS supplemented with 4% BSA, staining was performed with an anti-KSHV K8.1 antibody (sc-65446, Santa Cruz, 1:50 dilution) and an Alexa Fluor 488-conjugated goat-anti-mouse secondary antibody (A10667, Life Technologies, 1:4000 dilution) in PBS with 1% BSA and 40 U/mL RNase inhibitor, murine (NEB). K8.1+ and K8.1− cell subpopulations were analyzed on an LSRFortessa flow cytometer (BD Biosciences) and/or sorted on a FACSAriaIIIu cell sorter (BD Biosciences). FACS Diva and FlowJo software was used to analyze the flow cytometry data.

Phan H.V., van Gent M., Drayman N., Basu A., Gack M.U, & Tay S. (2021). High-throughput RNA sequencing of paraformaldehyde-fixed single cells. Nature Communications, 12, 5636.

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Comprehensive Antibody Panel for DNA Damage Response

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Rabbit polyclonal anti-XRCC4 was produced in our laboratory against the full-length recombinant human protein and rabbit polyclonal anti-Ligase IV was manufactured by Biotem (Le Rivier d'Apprieu, France) against a peptide. For immunoblotting, were used mouse monoclonal antibodies anti-β-Actin (clone AC-15, Ambion), PARP-1 (clone 4C10–5, BD Pharmingen), Ku80 (clone 111, ThermoScientific), Ligase III (clone 7, BD biosciences), Ligase I (clone 10H5, Genetex), Chk1 (clone G4, Santa-Cruz), Chk2 (clone B4, Santa-Cruz) and anti-DNA-PKcs (clone 18.2, Abcam), rabbit monoclonal antibody anti-γH2AX (Abcam) and rabbit polyclonal antibodies anti-DNA-PKcs PhSer-2056 (Abcam), KAP-1 (Abcam), KAP-1 PhSer-824 (Bethyl Laboratories), H2AX (Abcam), Chk1 PhSer-345 and Chk2 PhThr-68 (Cell Signaling). Peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibodies were from Jackson Immunoresearch Laboratories. For flow cytometry, mouse monoclonal anti-γH2AX primary antibody (clone JBW301, EMD Millipore) and AlexaFluor488-conjugated goat anti-mouse secondary antibody (Life technologies) were used.

Yuan Y., Britton S., Delteil C., Coates J., Jackson S.P., Barboule N., Frit P, & Calsou P. (2015). Single-stranded DNA oligomers stimulate error-prone alternative repair of DNA double-strand breaks through hijacking Ku protein. Nucleic Acids Research, 43(21), 10264-10276.

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CTLA4 Expression in Melanocytes and Melanoma

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Human melanocytes and melanoma cells were fixed with 4% PFA for 10 min
and permeabilized with 0.25% Triton X-100 in PBS, then incubated with a blocking
buffer (1% BSA, 22.52 mg/ml glycine in PBST) for 30 min at room temperature, which
was followed by overnight incubation with mouse anti-human-CTLA4 (1:100, BNI3, BD
Biosciences) antibody at 4°C. The control groups were only incubated with antibody
dilution buffer (1% BSA in PBST). After washing, the fixed cells on cover slips
were incubated with Alexa Fluor 488–conjugated goat anti-mouse secondary antibody
(1:400, Life Technology) for 1h at room temperature. The cover slips were mounted with
VECTASHIELD mounting medium with DAPI (Vector Laboratories Inc) overnight and imaged with
Leica TCS SP8 Confocal microscope at the specified magnification.

Mo X., Zhang H., Preston S., Martin K., Zhou B., Vadalia N., Gamero A.M., Soboloff J., Tempera I, & Zaidi M.R. (2017). Interferon-γ signaling in melanocytes and melanoma cells regulates expression of CTLA-4. Cancer research, 78(2), 436-450.

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KSHV Reactivation Quantification Protocol

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BC3 cells were mock-treated or treated with a final concentration of 20 ng/mL tetradecanoyl phorbol acetate (TPA, Millipore Sigma) for 36 hours to induce KSHV reactivation. Cells were pelleted by centrifugation at 300g for 3 min and fixed with 4% PFA in PBS for 15 min at room temperature followed by permeabilization with 0.1% (v/v) Triton X-100 in PBS with 1% (w/v) BSA for 15 min at room temperature. After washing and blocking the cells in PBS supplemented with 4% BSA, staining was performed with an anti-KSHV K8.1 antibody (sc-65446, Santa Cruz, 1:50 dilution) and an Alexa Fluor 488-conjugated goat-anti-mouse secondary antibody (A10667, Life Technologies, 1:4000 dilution) in PBS/1% BSA supplemented with RNase inhibitor, murine (NEB). K8.1-positive and negative cell populations were analyzed on an LSRFortessa flow cytometer (BD Biosciences) and/or sorted on a FACSAriaIIIu cell sorter (BD Biosciences).

Van Phan H., van Gent M., Drayman N., Basu A., Gack M.U, & Tay S. (2021). Fixed single-cell RNA sequencing for understanding virus infection and host response. bioRxiv.

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Dual Immunolabeling Protocol for Striatal Neurons

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PFA-fixed coronal sections of the striatum were treated with 0.5% citrate buffer in a 75°C water bath for 20 min to maximize antibody penetration into the tissue. Non-specific Fc-binding sites were blocked by incubation with 4.5% NGS for 1 h and sections were then incubated overnight at 4°C with anti-NeuN antibody (1:1000, mouse, Millipore). The sections were rinsed three times in PBS and incubated with anti-DCLK3 antibody (1:1000, rabbit, 21890002, Novus). Both primary antibodies were diluted in PBS supplemented with 3% NGS and 0.2% Triton™ X-100. We used the Tyramide Signal Amplification Kit (Life Technologies), according to the manufacturer’s protocol, for high-density DCLK3 labelling. For NeuN labelling, sections were rinsed three times in PBS and incubated for 1 h with Alexa Fluor® 488-conjugated goat anti-mouse secondary antibody (1:1000, Life Technologies). They were then rinsed again and incubated in 4′,6-diamidino-2-phenylindole (DAPI) diluted 1:2000 in PBS for 10 mins, before a final rinse in PBS and mounting.

Galvan L., Francelle L., Gaillard M.C., de Longprez L., Carrillo-de Sauvage M.A., Liot G., Cambon K., Stimmer L., Luccantoni S., Flament J., Valette J., de Chaldée M., Auregan G., Guillermier M., Joséphine C., Petit F., Jan C., Jarrige M., Dufour N., Bonvento G., Humbert S., Saudou F., Hantraye P., Merienne K., Bemelmans A.P., Perrier A.L., Déglon N, & Brouillet E. (2018). The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin. Brain, 141(5), 1434-1454.

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Immunofluorescence Staining of Melanoma Cells

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Melanoma cells were seeded at 3x10⁴ cells per well in 12-well plates on sterile cover slips. 24h post seeding attached cells were fixed and probed with primary antibody (S2 Table) and AlexaFluor488 conjugated goat-anti-mouse secondary antibody (Life Technologies, Mulgrave, Australia, 1:3000) and mounted with Prolong Antifade containing DAPI (Invitrogen, Carlsbad, USA).

Po J.W., Ma Y., Balakrishna B., Brungs D., Azimi F., de Souza P, & Becker T.M. (2019). Immunomagnetic isolation of circulating melanoma cells and detection of PD-L1 status. PLoS ONE, 14(2), e0211866.

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Evaluating Cell Surface Markers in HEK293T Cells

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HEK293T cells were transfected with 400 ng of pCMV3-PSGL-1, pCMV3-CD43, pCMV3-CD164, pCMV3-CD34, pCMV3-PODXL1, pCMV3-PODXL2, pCMV3-TIM-1, or pCMV3-Empty DNA. At 48 h post transfection, 0.5–1 million cells were stained with one of the following primary antibodies: mouse anti-PSGL1 antibody (KPL-1) (BD Pharmingen), mouse anti-CD43 antibody (1G10) (BD Biosciences), mouse anti-CD164 antibody (67D2) (Biolegend), mouse anti-CD34 antibody (563) (BD Biosciences), mouse anti-TIM-1 antibody (219211) (R & D Systems), mouse monoclonal anti-PODXL1 antibody (222328) (R & D systems), or mouse monoclonal anti-PODXL2 antibody (211816) (R & D Systems), followed by staining with Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (Invitrogen).

Dabbagh D., He S., Hetrick B., Chilin L., Andalibi A, & Wu Y. (2021). Identification of the SHREK Family of Proteins as Broad-Spectrum Host Antiviral Factors. Viruses, 13(5), 832.

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