Alexa 546

Manufactured by Jackson ImmunoResearch

Sourced in United States

Alexa 546 is a fluorescent dye that can be used in various biological applications. It has an excitation maximum at 554 nm and an emission maximum at 573 nm. Alexa 546 can be conjugated to various biomolecules, such as antibodies, proteins, or nucleic acids, to facilitate detection and visualization in experimental settings.

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Lab products found in correlation

Cyanine3 (cy3), by Jackson ImmunoResearch (3 mentions) Alexa 488, by Jackson ImmunoResearch (3 mentions) Alexa 647, by Jackson ImmunoResearch (2 mentions) Psd 95, by Thermo Fisher Scientific (1 mentions) Neuronal nuclei (neun), by Merck Group (1 mentions) Nestin, by Merck Group (1 mentions) Zen software, by Zeiss (1 mentions) Gad67, by Merck Group (1 mentions) Mowiol 4 88, by Merck Group (1 mentions) Tra 1 81, by BD (1 mentions) Lsm 700 confocal microscope, by Zeiss (1 mentions) Vimentin, by Santa Cruz Biotechnology (1 mentions) Ctip2, by Abcam (1 mentions) Satb2, by Abcam (1 mentions) Alexa 555, by Jackson ImmunoResearch (1 mentions) Ab21685, by Abcam (1 mentions) Ab6640, by Abcam (1 mentions) Ab150435, by Abcam (1 mentions) Ab300071, by Abcam (1 mentions) Vectashield hard set mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)

Close spelling variants of this product

Alexa 546 conjugate (2 citations) Alexa Fluor 546 (2 citations) Alexa Fluor 488 conjugated F(ab′)2 anti-human IgG (109-546-098, (2 citations) Alexa‐488 or 546‐conjugated secondary antibodies (2 citations) Anti-mouse Alexa-546 (2 citations) Alexa Fluor 546-conjugated donkey anti-goat (2 citations) Alexa-546-conjugated secondary antibodies (2 citations)

6 protocols using alexa 546

Immunofluorescence Characterization of Cell Cultures

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Cell cultures were fixed using 4% formaldehyde in phosphate-buffered saline. Primary antibodies were incubated overnight at 4 °C in labeling buffer containing 0.05 M Tris, 0.9% NaCl, 0.25% gelatin and 0.5% Triton-X-100 (pH 7.4). The following primary antibodies were used: SOX2, Nestin, MAP2, TBR1, GAD67, NeuN and glial fibrillary acidic protein (GFAP) (Merck-Millipore); FOXG1 (ProSci, Poway, CA, USA); Vimentin (Santa Cruz Biotechnology, Dallas, TX, USA); AFP (R&D Systems, Minneapolis, MN, USA); TRA-1-81 and Nanog (Beckton Dickinson, Franklin Lakes, NJ, USA); OCT4, BRN2, SATB2, CUX1, CUX2 and CTIP2 (Abcam, Cambridge, UK); Synapsin, MAP2 (Synaptic Systems, Göttingen, Germany); and PSD95 (Thermo Fisher Scientific). The following secondary antibodies were used: Alexa-488, Alexa-546, Alexa-555 and Cy3 antibodies (Jackson ImmunoResearch, West Grove, PA, USA). Samples were imbedded in Mowiol 4-88 (Sigma-Aldrich), after which confocal imaging was performed with a Zeiss LSM700 confocal microscope using ZEN software (Zeiss, Oberkochen, Germany).

Gunhanlar N., Shpak G., van der Kroeg M., Gouty-Colomer L.A., Munshi S.T., Lendemeijer B., Ghazvini M., Dupont C., Hoogendijk W.J., Gribnau J., de Vrij F.M, & Kushner S.A. (2017). A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells. Molecular Psychiatry, 23(5), 1336-1344.

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Immunofluorescence Staining of Cardiac Tissues

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Frozen slices of heart were used for immunofluorescence histochemical staining. Cryostat cardiac sections fixed with 4% paraformaldehyde were blocked in solution containing 10% FBS and 0.1% Triton X-100 for 1 h at 4 °C, followed by incubation with antibodies against WGA (GDP1020, Servicebio), GRP78 (ab21685, Abcam), eNOS (ab300071, Abcam), Serca2(ab150435, Abcam) or F4/80 (ab6640, Abcam) at 4 °C for 24 h. After washing and incubating with a fluorescent secondary antibody (Alexa 488 or Alexa 546, Jackson ImmunoResearch, Laboratories, West Grove, PA) at 4 °C for 2 h, the slices were mounted using Vectashield hard-set mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA).

Liu B., Shalamu A., Pei Z., Liu L., Wei Z., Qu Y., Song S., Luo W., Dong Z., Weng X, & Ge J. (2023). A novel mouse model of heart failure with preserved ejection fraction after chronic kidney disease induced by retinol through JAK/STAT pathway. International Journal of Biological Sciences, 19(12), 3661-3677.

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Quantifying Hippocampal Neurogenesis in Mice

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Mice were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffer. Brains were postfixed for 48 hours and sectioned on a vibratome (Leica Microsystems) and collected in cold PBS. To count the BrdU in the hippocampal dentate gyrus SGZ, free-floating sections were then incubated with primary antibody for BrdU (1:500; Fitzgerald) and biotin conjugated secondary (Jackson ImmunoResearch, West Grove, PA) along with Alexa Fluor 633 (Invitrogen) was used. Slices were mounted in Vectashield (Vector) and observed with a fluorescence microscope (Olympus Fluoview FV10i, Confocal Laser Scanning Microscope, Center Valley, PA) for labeling at × 10. To determine the number of BrdU+ cells in the SGZ, we counted every sixth section (6 sections per brain) and multiplied the result by 6.^{42 (link)} Representative slices from each group were separately double stained for doublecortin (DCX) and BrdU to determine whether BrdU+ cells were also DCX+. Free-floating sections were incubated with primary antibodies for Brdu (1:500) and DCX (1:300; Millipore). After primary incubation, the corresponding secondary antibody conjugated with Alexa Fluor 633 (Invitrogen) for DCX and biotin conjugated secondary (Jackson ImmunoResearch) along with Alexa 546 (Jackson ImmunoResearch) was used for Brdu.

Apkarian A.V., Mutso A.A., Centeno M.V., Kan L., Wu M., Levinstein M., Banisadr G., Gobeske K.T., Miller R.J., Radulovic J., Hen R, & Kessler J.A. (2016). Role of adult hippocampal neurogenesis in persistent pain. Pain, 157(2), 418-428.

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Immunostaining and Confocal Imaging Protocol

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Immunostaining and imaging were performed as previously described³⁶. Cells from three-dimensional cultures were fixed with 2% paraformaldehyde/PBS for 10 min, permeabilized in 0.5% Triton X-100/10% Goat serum/10% fish gelatin/PBS for 1 h and incubated overnight in primary antibodies. Primary antibodies used were as follows: PAR6B 1/200 (Santa Cruz #sc-67393), aPKCι 1/100 (BD Transduction #610175), PARD3B 1/100 (Santa Cruz #sc-398761), myc 1/100 (Origene # TA150121), GFP 1/500 (Abcam #ab13970), PTPN14 1/100 (R&D Systems #MAB4458), E-cadherin 1/200 (Cell Signaling #3195S), V5 1/200 (Thermo Fisher Scientific # R960-25), β1-integrin 1/200 (Abcam #ab30394), ZO-1 1/100 (Cell Signaling #8193S), YAP 1/100 (Cell Signaling #14074) and Phalloidin 1/100 (Invitrogen #A34055). The secondary antibodies conjugated to Alexa488, Alexa546, Alexa647, and Cy3 (Jackson ImmunoResearch Laboratories) were used at 1:750. DNA was detected with Hoechst dye 33258. Confocal imaging was performed using a LSM700 microscope from Zeiss with 20X/0.8NA or 40X/1.4NA objective lenses and processed using FIJI/ImageJ^{37 (link)} (version 1.53c, https://imagej.net/software/fiji/)³⁶.

Wang L.T., Proulx M.È., Kim A.D., Lelarge V, & McCaffrey L. (2021). A proximity proteomics screen in three-dimensional spheroid cultures identifies novel regulators of lumen formation. Scientific Reports, 11, 22807.

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Collagen I and Fibronectin Immunofluorescence

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Fibroblasts and MSCs were plated on glass coverslips in 24-well plates. For wells to be analyzed for collagen I, cells were treated with 0.2mM ascorbate (Sigma-Aldrich). Monolayers were fixed with 3.7% paraformaldehyde in PBS for 10 minutes, blocked with 1% bovine serum albumin in PBS for 1 hour, incubated with rabbit anti-collagen I or anti-fibronectin [35 (link)] for 1 h, and then incubated with anti-rabbit secondary antibody conjugated with Alexa 546 (Jackson ImmunoResearch Laboratories, West Grove, PA). Nonspecific rabbit anti-serum was used as a negative control. Coverslips were counterstained for DNA with 4′,6-diamidino-2-phenylindole (DAPI) and mounted with ProLong Gold anti-fade reagent (Thermo Fisher, Waltham, MA). Coverslips were then visualized by fluorescence microscopy with a Nikon Eclipse Ti inverted microscope and CoolSNAP HQ2 charge-coupled device camera (Photometrics) using the appropriate filters for Alexa 546.

Denu R.A., Nemcek S., Bloom D.D., Goodrich A.D., Kim J., Mosher D.F, & Hematti P. (2016). Fibroblasts and Mesenchymal Stromal/Stem Cells are Phenotypically Indistinguishable. Acta haematologica, 136(2), 85-97.

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Immunofluorescence Staining of Oocytes

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An antibody against CENP-C made in guinea pig was made by generating a clone expressing amino acids 502–939 (Genscript). This guinea pig anti-CENP-C was used at 1:1000. Additional primary antibodies were rat anti-CID (Active motif, 1:100), rabbit anti-CID (Active motif, 1:100), rabbit anti-SPC105R (1:4000) [69 (link)], rabbit anti-GFP (Invitrogen, 1:400), rat anti-HA (Roche, 1:50), mouse anti-C(3)G (1:500) [70 (link)], two mouse anti-ORB antibodies, 6H4 and 4H8 (1:100 for each) [71 (link)] and mouse anti-α tubulin DM1A conjugated directly to FITC (Sigma, 1:50). The secondary antibodies that were used were Cy3, Alexa 546, Alexa 633, or Alexa 647 from Jackson Immunoresearch Laboratories, and Alexa 488 from Invitrogen. The oocytes were stained with Hoechst 33342 at 1:10,000 (10 μg/ml). FISH probes were obtained from IDT where the X359 repeat was labeled with Alexa 594, the dodeca repeat was labeled with Cy5, and the AACAC repeat was labeled with Cy3.

Fellmeth J.E., Jang J., Persaud M., Sturm H., Changela N., Parikh A, & McKim K.S. (2023). A Dynamic population of prophase CENP-C is required for meiotic chromosome segregation. bioRxiv.

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