Alexa fluor 546

Manufactured by Thermo Fisher Scientific

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Alexa Fluor 546 is a fluorescent dye used in various life science applications. It has an absorption maximum at 556 nm and an emission maximum at 573 nm, making it suitable for detection in the orange-red region of the visible spectrum.

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529 protocols using alexa fluor 546

Immunofluorescence Analysis of Protein Localization

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Cells were fixed as above, permeabilised in 0.2% Triton X-100 and incubated with rabbit polyclonal anti-hEnsa (1/100), anti-hGreatwall (1/100) and monoclonal anti-BrDU. Antibodies were revealed with anti-Rabbit Alexa Fluor 546 (Life Technologies, A11035, 1/1000), anti-Mouse Alexa Fluor 546 (Life Technologies, A11018, 1/1000), anti-Rabbit Alexa Fluor 488 (Life Technologies, A11070, 1/1000) and anti-Mouse Alexa Fluor 488 (Life Technologies, A11017, 1/1000).
Alexa Fluor 546-conjugated or Alexa Fluor 488-conjugated goat anti-rabbit or mouse antibodies (Molecular Probes, Life Technologies). Cells were analysed as described previously^{35 (link)}. Images were taken with a Leica SP5 confocal microscope or with a MetaMorph-driven software (Molecular Devices, Sunnyvale, CA) wide-field Axioimager Z2 fluorescence microscope (Zeiss, Jena, Germany) with a PL APO ×63 or ×40 objectives (numerical aperture 1.32; Leica, Melville, NY) and a CoolSNAP HQ2 3 camera (Photometrics, Woburn, MA). For quantification experiments, cells were acquired using scan slide module and analysed with ImageJ. Images were then processed using Photoshop (Adobe, San Jose, CA).

Charrasse S., Gharbi-Ayachi A., Burgess A., Vera J., Hached K., Raynaud P., Schwob E., Lorca T, & Castro A. (2017). Ensa controls S-phase length by modulating Treslin levels. Nature Communications, 8, 206.

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Immunofluorescence Staining of M1 Macrophages

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Primary human monocyte-derived macrophages were polarized to “M1” phenotype on coverslips. The cells were fixed with 4% paraformaldehyde and then permeabilized in 0.2% Triton (Biorad)/PBS. Blocking buffer 2% BSA (Sigma-Aldrich) was added for 30 min. Rabbit polyclonal anti-SP140 antibody (dilution 1:200) (ab171141; Abcam) or mouse polyclonal anti-SP100 antibody (dilution 1:200) (ab167605; Abcam) were added for 2 h) followed by 2 h of secondary antibody, Polyclonal goat anti-Rabbit, Alexa Fluor546 (A-11035, Invitrogen) (dilution of 1:1000), or goat anti-mouse, Alexa Fluor546 (A-21123, Life Technologies) (dilution of 1:500), respectively. DAPI (Thermo Fisher) was used for nuclear detection.

Ghiboub M., Koster J., Craggs P.D., Li Yim A.Y., Shillings A., Hutchinson S., Bingham R.P., Gatfield K., Hageman I.L., Yao G., O’Keefe H.P., Coffin A., Patel A., Sloan L.A., Mitchell D.J., Hayhow T.G., Lunven L., Watson R.J., Blunt C.E., Harrison L.A., Bruton G., Kumar U., Hamer N., Spaull J.R., Zwijnenburg D.A., Welting O., Hakvoort T.B., te Velde A.A., van Limbergen J., Henneman P., Prinjha R.K., de Winther M.P., Harker N.R., Tough D.F, & de Jonge W.J. (2022). Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140. BMC Biology, 20, 182.

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Immunofluorescence Staining of Paraffin Sections

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Stainings were performed on formalin‐fixed paraffin‐embedded sections (3 μm). Sections were deparaffinized and rehydrated. For hematoxylin and eosin, Prussian blue, and Sirius red staining, sections were processed according to standard protocols. For immunofluorescence stainings, antigen retrieval was performed followed by blocking and primary antibody incubation at 4°C overnight. The following primary antibodies were used: rabbit anti‐mouse collagen IV (1:200, #2150‐1470, Bio‐Rad), guinea pig anti‐mouse insulin (1:50, ab7842, Abcam), isolectin‐B4 (1:100, Life Technologies, #132450) with antigen retrieval at pH 6 with citrate buffer, rabbit anti‐mouse CD31 (1:50, ab28364, Abcam) with antigen retrieval at pH 9 with citrate buffer. After washing, sections were incubated with secondary antibody (1:200) for 1 h at room temperature. The following secondary antibodies were used: goat anti‐rabbit Alexa Fluor 647 (Life Technologies, A21245), goat anti‐rabbit Alexa Fluor 546 (Life Technologies, A11035), and goat anti‐guinea pig Alexa Fluor 546 (Life Technologies, A11074).

Hasan S.S., Jabs M., Taylor J., Wiedmann L., Leibing T., Nordström V., Federico G., Roma L.P., Carlein C., Wolff G., Ekim‐Üstünel B., Brune M., Moll I., Tetzlaff F., Gröne H., Fleming T., Géraud C., Herzig S., Nawroth P.P, & Fischer A. (2020). Endothelial Notch signaling controls insulin transport in muscle. EMBO Molecular Medicine, 12(4), e09271.

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Immunoblotting and Immunofluorescence Antibody Protocols

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Primary antibodies used for immunoblotting and immunofluorescence were as follows: polyclonal anti-prion antibody ab3531 (#ab3531, Abcam, Cambridge, MA), prion protein monoclonal antibody clone SAF-84 (#189775, Cayman, Ann Arbor, MI), recombinant Anti-PrP Fab HuM-D18 (InPro Biotechnology, San Francisco, CA), polyclonal anti-Iba1 (#013-27691, FUJIFILM Wako Chemicals, Richmond, VA), polyclonal anti-GFAP (#AB5541, Millipore Sigma), polyclonal anti-MAP2 (#NB300-213, Novus Biologicals, Centennial, CO). The secondary antibodies used for immunoblotting were goat anti-rabbit IgG-HRP (#474-1506, KPL), goat anti-mouse IgG–HRP (#474-1806, KPL), and goat anti-human IgG, F(ab’)₂ fragment specific, peroxidase-conjugated (#31414, Pierce, Rockford, IL). The secondary antibodies for immunofluorescence were goat anti-mouse IgG antibody (Alexa Fluor 546, #A-11003, ThermoFisher Scientific), donkey anti-rabbit IgG antibody (Alexa Fluor 488, #A-21206, ThermoFisher Scientific), goat anti-rabbit IgG antibody (Alexa Fluor 546, #A-11010, ThermoFisher Scientific), and goat anti-chicken IgG antibody (Alexa Fluor 488, #A-11039, ThermoFisher Scientific).

Bocharova O., Makarava N., Pandit N.P., Molesworth K, & Baskakov I.V. (2024). Multiple steps of prion strain adaptation to a new host. Frontiers in Neuroscience, 18, 1329010.

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Immunofluorescence Staining of TNF-α and MCT4

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To examine cell types containing TNF-α and MCT4 immunoreactivity, double immunofluorescence staining was performed according to our published protocol (33 (link)). In brief, rabbit anti-TNF-α (cat. no. ab66579, dilution 1:500; Abcam), goat anti-Ionized calcium binding adaptor molecule 1 (Iba1; cat. no. ab5076, dilution 1:400; Abcam) for microglia and rabbit anti-MCT4 (cat. no. ab244385, dilution 1:100; Abcam) and mouse anti-GFAP (cat. no. MAB360, dilution 1:400; Abcam) for astrocytes were used. The sections were incubated in the mixture of the antisera overnight at 4°C, and the incubated sections were reacted in mixture of both donkey anti-rabbit IgG, Alexa Fluor488 (cat. no. A32790, dilution 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) and goat anti-mouse IgG, Alexa Fluor546 (cat. no. A-11030, dilution 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) and donkey anti-rabbit IgG, Alexa Fluor546 (cat. no. A10040, dilution 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) and goat anti-mouse IgG, or Alexa Fluor488 (cat. no. A-11001, dilution 1:500; Invitrogen; Thermo Fisher Scientific, Inc.) 2 h at room temperature. The immunoreaction was examined under confocal microscope (LSM510 META NLO; Carl Zeiss AG) at magnification, ×20 in the Korea Basic Science Institute Chuncheon Center.

Ohk T.G., Ahn J.H., Park Y.E., Lee T.K., Kim B., Lee J.C., Cho J.H., Park J.H., Won M.H, & Lee C.H. (2020). Comparison of neuronal death and expression of TNF-α and MCT4 in the gerbil hippocampal CA1 region induced by ischemia/reperfusion under hyperthermia to those under normothermia. Molecular Medicine Reports, 22(2), 1044-1052.

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Comprehensive Immunofluorescence Staining Protocol

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Primary antibodies: 1:250 mouse monoclonal anti-chymase (Abcam, #ab2377) 1:200 mouse monoclonal anti- tryptase (Abcam, #ab2378), 1:200 goat polyclonal anti-chymase (Abcam, #ab111239), 1:100 rat monoclonal anti-c-Kit (biotin) (Abcam, #ab25022), 1:300 rabbit polyclonal anti-collagen I (Abcam, #ab34710), 1:200 rabbit polyclonal anti- COX2 (Abcam, #ab15191), 1:200 mouse monoclonal anti-βIII-Tubulin (Millipore, #MAB1637), 1:100 rabbit polyclonal anti-SCF (Fisher Scientific, #PA520746), 1:400 mouse monoclonal anti-GFAP (Sigma, #G3893) and 1:100 rabbit polyclonal anti-CD45-PerCP (BioLegend, #103130). Secondary antibodies: 1:500 goat anti-rabbit- AlexaFluor488 or AlexaFluor546 (Thermo Fisher Scientific, #A11035 or #A11034), 1:500 goat anti-mouse-AlexaFluor488, AlexaFluor546 or AlexaFluor633 (Thermo Fisher Scientific, #A11029, #A11030, or #A21052), 1:500 donkey anti-goat- AlexaFluor488 (Thermo Fisher Scientific, #A11055) and 1:500 Streptavidin- AlexaFluor633 (Thermo Fisher Scientific, #S21375). NeuroTrace 530/615 red fluorescent Nissl stain (Thermo Fisher Scientific, #B34650) was also used for neuronal visualization.

Kovacs M., Alamón C., Maciel C., Varela V., Ibarburu S., Tarragó L., King P.H., Si Y., Kwon Y., Hermine O., Barbeito L, & Trias E. (2021). The pathogenic role of c-Kit+ mast cells in the spinal motor neuron-vascular niche in ALS. Acta Neuropathologica Communications, 9, 136.

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Immunofluorescence Analysis of Podocyte Tight Junctions

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For immunofluorescence experiments, PTL were differentiated in CellCarrier ultra-black 96-well plates. On day 14 of differentiation, PTL were fixed with 4% PFA (20 min), permeabilized with 0.1% Triton X-100 (10 min), and blocked with 5% BSA (1 h). Primary antibodies were applied for 1.5 h at RT in a DPBS 1% BSA solution and include megalin/LRP2 (1:100) (R&D systems MAB9578), ZO-3 (1:1600) (Cell Signaling Technology 3704), and occludin Alexa Fluor 594 (1:250) (ThermoFisher Scientific 331594). Secondary antibodies were applied (when applicable) together with and Hoechst 33342 (1:10.000) (ThermoFisher H3570) for 1 h at RT in a DPBS 1% BSA solution and include α-rabbit Alexa FluorTM 546 (1:1000) (ThermoFisher A10040) and α-mouse Alexa FluorTM 546 (1:1000) (ThermoFisher A10036). Cells were imaged using the Operetta CLS High-Content Imager (Perkin Elmer) with 40 × water objective, and images were exported using the Harmony software 4.8.

Jennings P., Carta G., Singh P., da Costa Pereira D., Feher A., Dinnyes A., Exner T.E, & Wilmes A. (2022). Capturing time-dependent activation of genes and stress-response pathways using transcriptomics in iPSC-derived renal proximal tubule cells. Cell Biology and Toxicology, 39(4), 1773-1793.

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Visualizing HPV Oncoprotein Expression

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The 293FT, UM104 and Caski cells (2 × 10E5) were seeded in 35 mm glass-bottom dishes (MatTek Corporation). After 24 h, these cells were transfected with pCAGIP-PA-E6 or pCAGIP-PA-E7 using Fugene HD and cultured for 48 h. After washing with PBS, these cells were fixed with 4% paraformaldehyde (PFA) for 5 min at room temperature. After permeabilisation of these cells with 0.5% Triton-X 100 for 10 min at room temperature, these cells were blocked with 5% skimmed milk for 30 min at room temperature, and then incubated with the primary antibodies: anti-E7 (ab20191, Abcam, 1:100 dilution; sc-6981, Santa Cruz, 1:50 dilution), anti-PA tag (NZ-1, Wako, 1:100 dilution), or anti-mouse control IgG (ab18413, Abcam, 1:100 dilution) at 4 °C overnight. The goat anti-mouse IgG conjugated with Alexa Fluor 546 (Invitrogen) was used as a secondary antibody for E7 and the mouse control IgG or goat anti-rat IgG conjugated with Alexa Fluor 546 (Invitrogen) were used as secondary antibodies for the PA tag. The pictures of the immunofluorescently stained cells were captured with a confocal laser microscope (A1Rsi/Ti-PF, Nikon).

Kitamura K., Nimura K., Ito R., Saga K., Inohara H, & Kaneda Y. (2020). Evaluation of HPV16 E7 expression in head and neck carcinoma cell lines and clinical specimens. Scientific Reports, 10, 22138.

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Immunofluorescence of Embryonic Cardiac Markers

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E11.5, E12.5 and E14.5 embryos were fixed with 4% PFA in PBS at 4 °C overnight. After cryoprotection in 10 and 30% sucrose, the tissue was embedded and frozen in OCT. Eight micrometer sections were used for IF. Slides were briefly fixed with 4% PFA, washed and permeabilized with PBS containing 0.1% Triton X-100 at room temperature for three times, 5 min each. After blocking with 10% normal donkey serum and 1% BSA at room temperature for 1 h, sections were incubated in rabbit anti-p-IGF-IR (Santa Cruz SC101703) and goat anti-Troponin C (Abcam ab30807), or goat anti-p-Insulin receptor (pINSR) (SC25103) and rabbit anti- MYL2 (Abcam ab79935) in PBS containing 1% BSA and 10% donkey serum at 4 °C overnight. Secondary antibodies donkey anti-rabbit (Invitrogen Alexa Fluor 546) and donkey anti-goat (Invitrogen Alexa Fluor 488), or donkey anti-goat (Invitrogen Alexa Fluor 546) and donkey anti-rabbit (Invitrogen Alexa Fluor 488) were used to detect pIGF1R and Troponin C, or pINSR and MYL2 with 1 h incubation at room temperature. Slides were mounted with mounting media containing DAPI.

Wang K., Shen H., Gan P., Cavallero S., Kumar S.R., Lien C.L, & Sucov H.M. (2019). Differential roles of insulin like growth factor 1 receptor and insulin receptor during embryonic heart development. BMC Developmental Biology, 19, 5.

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AQP4 and Plectin Immunolabeling in NMO

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Cells plated onto glass coverslips coated with PDL (1:100) and laminin (1:100) were labeled as described [38 (link), 39 (link)]. Briefly, cells were incubated with 3% BSA in 1 × PBS to prevent nonspecific background signal (37 °C, 1 h). Then, to label AQP4 in the plasmalemma, heat-inactivated NMO-IgG patient serum (1:100) was applied (+ 4 °C, 15 min). The AQP4 antibodies in NMO-IgG serum exhibit a high affinity for the extracellular surface of AQP4 [40 (link)]. To label surface plectin, rabbit antiserum to plectin (#46, 1:100) was applied (+ 4 °C, 15 min). Next, cells were fixed in 2% formaldehyde (RT, 15 min), incubated with the appropriate secondary antibodies (37 °C, 45 min), goat anti-human IgG (Alexa Fluor 546, 1:600; Invitrogen), donkey anti-human IgG (Abberior Star Green, 1:600; Abberior, Göttingen, Germany), or goat anti-rabbit IgG (Alexa Fluor 488 or Alexa Fluor 546, 1:600; Invitrogen) and mounted onto glass slides using Mowiol 4–88 mounting medium.

Žugec M., Furlani B., Castañon M.J., Rituper B., Fischer I., Broggi G., Caltabiano R., Barbagallo G.M., Di Rosa M., Tibullo D., Parenti R., Vicario N., Simčič S., Pozo Devoto V.M., Stokin G.B., Wiche G., Jorgačevski J., Zorec R, & Potokar M. (2024). Plectin plays a role in the migration and volume regulation of astrocytes: a potential biomarker of glioblastoma. Journal of Biomedical Science, 31, 14.

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