Alexa fluor 594 labeled goat anti rabbit igg

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Alexa Fluor 594-labeled goat anti-rabbit IgG is a fluorescently-labeled secondary antibody used for detection of rabbit primary antibodies in various immunoassay techniques. It contains a goat-derived antibody directed against rabbit immunoglobulin G (IgG) and is conjugated to the Alexa Fluor 594 fluorescent dye.

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16 protocols using alexa fluor 594 labeled goat anti rabbit igg

Immunofluorescent Staining of Cells

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Cells were fixed in ice-cold methanol for 20 min at −20 °C, rehydrated three times with phosphate-buffered saline (PBS), and blocked in 5% bovine serum albumin–PBS for 30 min. The cells were stained with primary antibody in blocking buffer for 1 h at 37 °C, rinsed in PBS, and stained again with Alexa Fluor 488-labeled rabbit anti-mouse IgG or Alexa Fluor 594-labeled goat anti-rabbit IgG (Life Technologies) for 1 h at 37 °C. The cells were then rinsed with PBS containing 4′, 6-diamidino-2-phenylindole (DAPI) and mounted. The cells were observed under a confocal microscope (LSM 710 NLO and DuoScan System, Zeiss).

Chen H.W., Yang Y.K., Xu H., Yang W.W., Zhai Z.H, & Chen D.Y. (2015). Ring finger protein 166 potentiates RNA virus-induced interferon-β production via enhancing the ubiquitination of TRAF3 and TRAF6. Scientific Reports, 5, 14770.

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Immunofluorescence Staining of GCPs

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The tissue samples were stained with anti-LacZ, anti-caspase 3, or anti-Slc7a11 as described elsewhere^{67 (link),68 (link)}. GCPs were fixed in 4% formaldehyde for 10 min, washed with PBS, incubated with a blocking solution containing 10% fetal bovine serum and 0.1% Triton X-100 in PBS for 15 min to block non-specific binding sites, and incubated overnight with the anti-Atoh1 (1:400; Chemicon; AB5692), anti-LacZ antibody, or anti-Slc7a11 primary antibodies. After washing with PBS-Tween, the cells were incubated for 1.5 h with Alexa Fluor594-labeled goat anti-rabbit IgG (Life Technologies, Grand Island, NY, USA; A11037) and processed using 4′,6-diamino-2-phenylindole to visualize cell nuclei (1:3000; 5 mg/ml stock in dimethyl sulfoxide, Merck). Cells were mounted on slides using ProLong Gold Antifade Mountant (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA; P36934), and cell immunofluorescence was visualized using an Olympus FV1000 confocal laser scanning microscope.

Ku C.C., Wuputra K., Kato K., Lin W.H., Pan J.B., Tsai S.C., Kuo C.J., Lee K.H., Lee Y.L., Lin Y.C., Saito S., Noguchi M., Nakamura Y., Miyoshi H., Eckner R., Nagata K., Wu D.C., Lin C.S, & Yokoyama K.K. (2020). Jdp2-deficient granule cell progenitors in the cerebellum are resistant to ROS-mediated apoptosis through xCT/Slc7a11 activation. Scientific Reports, 10, 4933.

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Astrocyte Identification in Autoimmunity

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To identify astrocytes as a possible target of the autoimmune reaction, double-labeling immunofluorescence was performed. A fluorescein isothiocyanate-conjugated sheep polyclonal antibody against canine immunoglobulin G (IgG, 1 : 250; Bethyl Laboratories, USA) and a rabbit polyclonal antibody against human GFAP (1 : 1000; Abcam, USA) were used as primary antibodies for staining the antigen-retrieved slides. After three washes with PBS, the sections were incubated with secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG; Life Technologies, USA) at room temperature for 1 h. The slides were then mounted with Vectashield medium containing 4',6-diamidino-2-phenylindole (DAPI; Vector Labs, USA). Colocalization studies were performed with a fluorescence microscope (DM6000B; Leica Microsystems, Germany).

Moon J.H., Jung H.W., Lee H.C., Jeon J.H., Kim N.H., Sur J.H., Ha J, & Jung D.I. (2015). A study of experimental autoimmune encephalomyelitis in dogs as a disease model for canine necrotizing encephalitis. Journal of Veterinary Science, 16(2), 203-211.

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Osteocyte Immunofluorescence Imaging Protocol

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WT and Sost KO osteocytes were grown on 12-well, collagen treated, glass bottom plates (MatTek Corporation). Cells were fixed in 4% PFA, washed in PBS, and blocked in 10% goat serum in PBS. The following primary antibodies were used: podoplanin (8.1.1, Santa Cruz Biotechnology, 1:50 dilution), Lef-1 (N-17, Santa Cruz Biotechnology, 1:50 dilution), β-catenin (Cell Signaling Technologies, 1:1000 dilution). After several PBS washes, secondary antibody was added. For podoplanin: Alexa Fluor 488-labeled goat anti-hamster IgG, 1:200 dilution; for Lef-1: Alexa Fluor 488-labeled donkey anti-goat IgG, 1:200 dilution; for β-catenin: AlexaFluor 594-labeled goat anti-rabbit IgG, 1:200 dilution (all from Life Technologies). Cells were washed with PBS, and counterstained with Vectashield Hard Set Mounting Medium containing 4’,6-diamidino-2-phenylindole (Vector Laboratories). Cells were photographed on an Axio Observer inverted microscope; fluorescence micrographs were taken using a Zeiss Super Resolution Elyra microscope system with Structured Illumination and a Plan-Apochromat 63X/1.4NA oil objective controlled by ZEN 2010 software.

Ryan Z.C., Craig T.A., Salisbury J.L., Carpio L.R., McGee-Lawrence M., Westendorf J.J, & Kumar R. (2014). Enhanced Prostacyclin Formation and Wnt Signaling in Sclerostin Deficient Osteocytes and Bone. Biochemical and biophysical research communications, 448(1), 83-88.

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Double Immunofluorescence Staining Protocol

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The procedure used was similar to that for immunohistochemistry, but without incubation in 1% H₂O₂. The primary antibodies used for the immunofluorescence procedure were the same as those already described (IBA1, Wako, 1:1000). Sections were first incubated with the primary antibody overnight at 4 °C. The next day, they were incubated with a fluorescent secondary antibody (Alexa Fluor 594-labeled goat anti-rabbit IgG or Alexa Fluor 488-labeled goat anti-rabbit IgG (1:1000, Life Technologies)) for 1 h at RT. Sections were then washed and incubated overnight at 4 °C with another primary antibody. Finally, they were incubated with a second fluorescent secondary antibody (Alexa Fluor 488-labeled goat anti-mouse IgG or 594-labeled goat anti-mouse IgG (1:1000, Life Technologies)) for 1 h at RT. The sections were stained with DAPI, washed, and mounted in a fluorescence mounting medium. Images were acquired with a laser confocal microscope (SP8, Leica, Wetzlar, Germany) or an epifluorescence microscope (DM6000, Leica, Wetzlar, Germany).

Cresto N., Gardier C., Gaillard M.C., Gubinelli F., Roost P., Molina D., Josephine C., Dufour N., Auregan G., Guillermier M., Bernier S., Jan C., Gipchtein P., Hantraye P., Chartier-Harlin M.C., Bonvento G., Van Camp N., Taymans J.M., Cambon K., Liot G., Bemelmans A.P, & Brouillet E. (2021). The C-Terminal Domain of LRRK2 with the G2019S Substitution Increases Mutant A53T α-Synuclein Toxicity in Dopaminergic Neurons In Vivo. International Journal of Molecular Sciences, 22(13), 6760.

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Quantitative Analysis of AcH3K9 in Parthenogenetic Blastocysts

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For day 7 embryos derived from parthenogenetically activated (PA), zonae pellucidae were removed by pronase treatment. Blastocysts were washed in PBS, then fixed for 15 min in 4% paraformaldehyde in PBS, and embryos were permeabilized with 0.2% Triton X-100 in PBS-PVA for 30 min. Samples were blocked overnight at 4ºC in blocking solution. The samples were incubated with a rabbit polyclonal antibody to AcH3K9 (Millipore, Darmstadt, Germany ) diluted 1:200 for 12 h at 4ºC. After extensive washing, samples were incubated with a secondary antibody of Alexa Fluor 594-labeled goat anti-rabbit IgG (Invitrogen, Eugene, Oregon, USA) for AcH3K9. After washing three times, blastocysts were stained with Hoechst 33342 and mounted on slides. Fluorescence was observed under a fluorescence digital microscope (Axiovert 200M, ZEISS, Shanghai, China), and the fluorescence intensity of each pixel was transformed to obtain a quantitative measure using Image-J software. Quantification of AcH3K9 signal intensities in BCB+ and BCB− groups was expressed relative to that of the control embryos (set as 100%). Quantification of the AcH3K9 is represented as the mean±standard error (SE). Values with different superscripts means significant difference (p<0.05).

Fu B., Ren L., Liu D., Ma J.Z., An T.Z., Yang X.Q., Ma H., Zhang D.J., Guo Z.H., Guo Y.Y., Zhu M, & Bai J. (2015). Subcellular Characterization of Porcine Oocytes with Different Glucose-6-phosphate Dehydrogenase Activities. Asian-Australasian Journal of Animal Sciences, 28(12), 1703-1712.

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Immunofluorescent Labeling of LV Myocytes

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Freshly isolated WT mouse LV myocytes adherent to laminin-coated coverslips were washed 3x with phosphate buffered saline containing 2 mM EGTA (PBS-E). Adult LV myocytes and neonatal ventricular myocytes were fixed for 30 min in 4% paraformaldehyde in PBS-E. After 2 rinses with PBS-E, myocytes were permeabilized for 2 min with 0.05% Triton X-100. Myocytes were rinsed 2x with PBS-E, and once with BLOTTO (5% nonfat dry milk, 0.1 M NaCl, and 50 mM Tris.HCl; pH 7.4). Primary antibodies against BAG3 (1:50; Proteintech Group Inc, Chicago, IL) diluted in BLOTTO were added to the cells, incubated at room temperature in the dark for 60 min, and rinsed 3x with BLOTTO. Secondary antibodies (Alexafluor 594-labeled goat anti-rabbit IgG; 1:50; Invitrogen, Eugene, OR) diluted in BLOTTO were added to the cells, incubated in the dark for 30 min, and followed by 3 PBS-E rinses. Na⁺-K⁺-ATPase was detected with Alexafluor 488-labeled anti-α1 subunit antibody (1:250; Millipore; ex. 488 nm, em. 510 nm). L-type Ca²⁺ channels were detected with anti-α1c-Ca_v1.2 antibody (1:250, Millipore; secondary antibody Alexafluor 488-labeled goat anti-rabbit IgG). Coverslips were mounted to slides with Prolong Gold Anti-fade mounting solution (Invitrogen). Confocal images (63× oil objective; 510 Meta; Carl Zeiss, Inc.) were acquired at 594 nm excitation and 617 nm emission for BAG3.

Feldman A.M., Gordon J., Wang J., Song J., Zhang X.Q., Myers V.D., Tilley D.G., Gao E., Hoffman N.E., Tomar D., Madesh M., Rabinowitz J., Koch W.J., Su F., Khalili K, & Cheung J.Y. (2016). BAG3 regulates contractility and Ca2+ homeostasis in adult mouse ventricular myocytes. Journal of molecular and cellular cardiology, 92, 10-20.

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Immunofluorescence Staining of Fibrotic Liver Tissues

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Immunofluorescence staining was performed on fibrotic tissues collected from the mice livers post injection of ProCA32 and ProCA32.collagen1 at 24 h time point. Cryosections of fibrotic liver tissues were cut at a thickness of 5 μm. The tissues were fixed with 4% (vol/vol) formaldehyde for 10 min at room temperature and then washed three times with Tris-buffered saline with Tween 20 (TBST). Tissues were then blocked at room temperature for 1 h with 1% bovine serum albumin (BSA) in TBST, and then incubated with two primary antibodies for 1.5 h. A ProCA32.collagen1 rabbit antibody prepared by our lab was used as a primary antibody for the contrast agent at 1:100 dilution. Another primary antibody for collagen type I, Anti-Collagen I antibody [COL-1] (ab34710) was used with 1:500 dilution. After washing three times with TBST, the slides were incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (Invitrogen; catalog #R37116) as a secondary antibody for collagen type I with 1:200 dilution. Alexa Fluor 594-labeled goat anti-rabbit IgG (Invitrogen; catalog #A-11012) was used as a secondary antibody for ProCA32.collagen1 (1:200 dilution). The nuclei of the cells were stained by DAPI (blue, ThermoFisher scientific, catalog #62248, 1:1000 dilution).

Salarian M., Turaga R.C., Xue S., Nezafati M., Hekmatyar K., Qiao J., Zhang Y., Tan S., Ibhagui O.Y., Hai Y., Li J., Mukkavilli R., Sharma M., Mittal P., Min X., Keilholz S., Yu L., Qin G., Farris A.B., Liu Z.R, & Yang J.J. (2019). Early detection and staging of chronic liver diseases with a protein MRI contrast agent. Nature Communications, 10, 4777.

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Immunofluorescence Characterization of hiPSC-CMs

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Well-suspended hiPSC cells and induced cardiomyocytes were seeded in a μ-Slide 8 well (80827, ibidi, USA) pre-coated with Matrigel (1：100) at the density of 2×10⁴ per well. 48 h later cells were fixed with 4% (w/v) Paraformaldehyde (PFA) for 15 min at room temperature, permeabilized, and incubated with following first primary antibodies: anti-OCT4 antibody (#2750, Cell Signaling Technology, USA), anti-NANOG antibody (#3580, Cell Signaling Technology, USA), anti-SSEA4 antibody (#4755, Cell Signaling Technology, USA), anti-TRA-1-60 antibody (#4746, Cell Signaling Technology, USA), anti-MKI67 antibody (ab15580, abcam, USA), anti-NKX2-5 antibody (ab91196, abcam, USA), anti-cTNT antibody (MS-295-P1, Thermo Fisher Scientific, USA) and anti-α-ACTININ antibody (A7811, Sigma, USA), followed by their corresponding fluorescence-labeled secondary antibodies, alexa fluor 488 labeled goat anti-rabbit IgG (A-11008, Invitrogen, USA), alexa fluor 488 labeled goat anti-mouse IgG (A-11001, Invitrogen, USA), alexa fluor 594 labeled goat anti-rabbit IgG (R37177, Invitrogen, USA) and alexa fluor 594 labeled goat anti-mouse IgG (A-11005, Invitrogen, USA). The slides were then counterstained using 0.5 μg/ml of DAPI (#4083, Cell Signaling Technology, USA) for 15 min at room temperature. After rinsing with PBS, the chambers were mounted and visualized by fluorescence microscopy (IX83, Olympus, Japan).

Liu Y., Zhang P., Huang W., Liu F., Long D., Peng W., Dang X., Zeng X, & Zhou R. (2021). p53 Promotes Differentiation of Cardiomyocytes from hiPSC through Wnt Signaling-Mediated Mesendodermal Differentiation. International Journal of Stem Cells, 14(4), 410-422.

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Immunostaining of Liver Sections for Plasmodium vivax Infection

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FRG huHep mice were intravenously injected with one million P. vivax sporozoites. Livers were harvested on day 8 post-infection and fixed in 10% formalin for 24 h before they were sliced to 50 μm sections with a vibratome. Liver sections were permeabilized in 10% H₂O₂ and 0.25% Triton X-100 in 1× TBS for 30 min at room temperature with agitation, then washed in 1× TBS and blocked in 5% milk in 1× TBS for 1 h at room temperature. The primary antibodies, α-rUIS4 mAb and HSP60 mAb, were diluted to 1:500 in 5% milk in 1× TBS and liver sections were incubated over night at 4 °C with agitation. After primary antibody staining, liver sections were washed five times in 1× TBS for 5 min per wash before addition of 2 μg/mL Alexa Fluor 488-labeled goat anti-mouse IgG (Thermo Fisher) and Alexa Fluor 594-labeled goat anti-rabbit IgG (Thermo Fisher) for 2 h at room temperature. Liver sections were again washed five times for 5 min per wash before they were transferred to 1 mL 0.06% KMnO₄ in H₂O. After another wash in 1× TBS, cells were stained in 2 μg/mL DAPI for 5 min at room temperature. After washing in 1× TBS, sections were mounted using Prolong Gold Antifade reagent to preserve fluorescence.

Schafer C., Dambrauskas N., Steel R.W., Carbonetti S., Chuenchob V., Flannery E.L., Vigdorovich V., Oliver B.G., Roobsoong W., Maher S.P., Kyle D., Sattabongkot J., Kappe S.H., Mikolajczak S.A, & Sather D.N. (2018). A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages. Malaria Journal, 17, 370.

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