Goat anti rabbit alexa 594

Manufactured by Thermo Fisher Scientific

Sourced in United States

Goat anti-rabbit Alexa 594 is a secondary antibody conjugated with the Alexa Fluor 594 dye. It is designed to bind and detect rabbit primary antibodies, allowing for fluorescent labeling and visualization of target proteins or molecules in various applications such as immunohistochemistry, flow cytometry, and Western blotting.

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Alexa 594 (538 citations) Alexa Fluor 594 goat anti-rabbit secondary antibody (40 citations) Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (17 citations) Alexa 594-conjugated goat anti-rabbit secondary antibody (10 citations) Alexa Flour 594 goat anti-rabbit IgG (7 citations) Alexa Flour 594 goat anti-rabbit (6 citations) Alexa Fluor 594 goat anti-mouse or rabbit IgG (5 citations) Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody Alexa Fluor™ 594 (4 citations) Alexa Fluor® 594 goat anti-rabbit IgG (H+L) antibody (4 citations) Alexa 488- or 594-conjugated goat anti-rabbit (3 citations)

35 protocols using goat anti rabbit alexa 594

Immunofluorescence Staining of FFPE Brain Tissue

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Formalin-Fixed Paraffin-Embedded (FFPE) human brain tissue was sectioned to 5 μm and mounted on microscope slides. The sections were dewaxed at 60 °C for 30 min and then transferred into HistoChoice (Sigma) for 2 washes for 5 min each. Sections were then moved into 100%, 95% and 70% ethanol for rehydration followed by three washes in PBS. For C3 staining, sections underwent antigen retrieval in M6 buffer (2.1% citric acid monohydrate, 2.94% tri-sodium citrate in dH₂O, pH 6) at 95 °C for 10 min.
Sections were then blocked in blocking buffer containing 10% normal goat serum (NGS), 0.4% Triton X-100 (Sigma) in PBS for 1h at room temperature. The following primary antibodies were used: C3d (Dako A0063, rabbit 1:600), GFAP (Dako Z0034, rabbit, 1:500), GFAP (Sigma G3893, mouse, 1:400) and SPARC (R&D MAB941, mouse, 25 ug/mL). Sections were incubated in the primary antibodies over night at 4 °C in blocking buffer followed by three washes in PBS. Sections were then incubated in the following secondary antibodies: goat anti-rabbit Alexa 594 (Invitrogen) and goat anti-mouse Alexa 488 (Abeam) at room temperature for 1h. Sections were then washed in PBS, incubated in TrueBlack (biotium) for 1 min, counter-stained with DAPI and mounted using Fluoromount-G (SouthernBiotech). All images were acquired on a Keyence BZ-X710 using a 20x objective and processed in Fiji.

Sadick J.S., O’Dea M.R., Hasel P., Dykstra T., Faustin A, & Liddelow S.A. (2022). Astrocytes and oligodendrocytes undergo subtype-specific transcriptional changes in Alzheimer’s disease. Neuron, 110(11), 1788-1805.e10.

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Spinal Cord Tissue Processing and Axon Visualization

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Segments of spinal cord containing the PNGs were dissected and post-fixed overnight in 4% PFA at 4°C. The tissue then was cryoprotected in 30% sucrose before sectioning on a cryostat. To visualize BDA-labeled axons in the animals injected with BDA, horizontal sections (25 μm) were cut in a series of six sets. Sections were blocked in 5% normal goat serum, 1% bovine serum albumin, 0.1% Triton X-100 in PBS for 1 hour at room temperature and then incubated with anti-GFAP (Dako) overnight at 4°C. The sections were rinsed in PBS and incubated in streptavidin-Alexa 488 or goat anti-rabbit Alexa 594 (Invitrogen) overnight at 4°C. Sections were rinsed in PBS, mounted onto glass slides, and coverslipped using FluorSave (Calbiochem).
For animals that were electrically stimulated, transverse sections of spinal cord within 1.5mm caudal to the distal interface was sectioned in a series of six, blocked in the same blocking solution described above, and incubated with an antibody to c-Fos (Abcam, Cambridge, MA). The sections were rinsed and incubated with goat-anti rabbit Alexa 594 overnight at 4°C. Sections were rinsed in PBS, mounted onto glass slides, and coverslipped using FluorSave (Calbiochem).
All sections were imaged using an Olympus BX51 microscope.

Xu C., Klaw M.C., Lemay M.A., Baas P.W, & Tom V.J. (2014). Pharmacologically inhibiting kinesin-5 activity with monastrol promotes axonal regeneration following spinal cord injury. Experimental neurology, 263, 172-176.

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Immunostaining of Neuronal Cytoskeleton

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Sections were briefly rinsed in 1xTBS, followed by an incubation in 10% methanol and 3–4% H₂O₂ in 1xTBS, before the sections were rinsed in 1xTBS and incubated overnight in 4°C in blocking buffer with 0.5% gelatin and 0.01% Triton-X in TBS supplied the primary antibody for anti β-tubulin 1:500 rabbit (Biosite) or anti red fluorescent protein (RFP) rabbit 1:200 (Abcam). The sections were then rinsed repeatedly in 1xTBS followed by incubation for 1 h in RT in blocking buffer with 0.5% gelatin and 0.01% Triton-X in TBS supplied with secondary antibody goat anti-rabbit Alexa 594 1:800 (Invitrogen) or anti-rabbit Alexa 647 1:400 (Invitrogen).

Rogoz K., Stjärne L., Kullander K, & Lagerström M.C. (2015). VGLUT2 controls heat and punctuate hyperalgesia associated with nerve injury via TRPV1-Cre primary afferents. PLoS ONE, 10(1), e0116568.

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Immunostaining of Drosophila Embryos

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Embryos of varying H2Av dosage were obtained as described above. Embryos were collected on apple juice agar plates, aged appropriately, dechorionated in 50% bleach, and fixed in 4% formaldehyde/1xPBS for 20 min. Embryos were then de-vitellinized using heptane/methanol and washed extensively in 1x PBS/0.1% Triton X-100 (Sigma). Embryos (equal number of embryos per tube) were incubated in rabbit anti-H2AvD antibody (Active Motif, Carlsbad, CA) at 1:1000 final concentration O/N at 4°C. Embryos were washed extensively in 1x PBS/0.1% Triton X-100 and incubated for 4 hr at RT in goat anti-rabbit Alexa 594 (Invitrogen) at a 1:1000 final concentration. Embryos were washed extensively and mounted in Vectashield (Vectorlabs, Burlington, ON, CAN) for imaging.

Johnson M.R., Stephenson R.A., Ghaemmaghami S, & Welte M.A. (2018). Developmentally regulated H2Av buffering via dynamic sequestration to lipid droplets in Drosophila embryos. eLife, 7, e36021.

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Immunolabeling of Synaptic Proteins

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Primary antibodies used were as follows: rat anti-HA 1:100 (clone 3F10), mouse anti-GFP (clones 7.1 and 13.1) (Roche Diagnostics); mouse anti-Synapsin 1:2000 (clone 46.1), rabbit anti-PSD-95 1:1000 (Synaptic Systems, Goettingen, Germany). Secondary antibodies used were as follows: goat anti-rat Alexa 594 (1:4,000) and anti-rat Alexa 647 (1:400), goat anti-mouse Alexa 488 (1:2,000), and Alexa 350 (1:500); goat anti-rabbit Alexa 594 (1:4,000) (all from Invitrogen); goat anti-mouse Abberior STAR 440SX (Abberior GmbH, Göttingen, Germany).

Stanika R., Campiglio M., Pinggera A., Lee A., Striessnig J., Flucher B.E, & Obermair G.J. (2016). Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology. Scientific Reports, 6, 34528.

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Immunocytochemical Staining of Cell Populations

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FACs-sorted cells were suspended in PBS⁺ 2% FBS at a concentration of 5 × 10⁴ cells/ml, and 200 µl of cell suspension was spun onto each glass slide using a cytocentrifuge at 800 rpm for 5 min. Slides were air dried, fixed in 100% ice-cold acetone, and air dried again. Cells were permeabilized in Tris-buffered saline (TBS) containing 0.1% Tween for CK and vimentin or 0.3% Triton X-100 for p53 (TBS-T) for 10 min and then incubated for 30 min in TBS-T/5% BSA/5% goat serum, followed by incubation with the following antibodies: rabbit anti-wide spectrum CK (Abcam; 1:100), mouse-anti-human vimentin (Abcam; 1:100), mouse-anti-human p53 (Santa Cruz; 1:100) overnight at 4°C. The following day, slides were washed three times in TBS and incubated with the appropriate secondary antibodies: goat-anti-rabbit-Alexa594 (Invitrogen; 1:1,000) plus goat-anti-mouse-Alexa488 (Invitrogen; 1:400) for pan-CK and vimentin; or goat-anti-mouse-Alexa594 (Invitrogen; 1:200) for p53. Secondaries were incubated for 1 h at room temperature. Slides were then washed and incubated for 1 min in TBS with 1 µg/ml Hoechst 333258, then coverslipped with Mowiol 4–88 (Sigma-Aldrich).

Hussain A., Voisin V., Poon S., Karamboulas C., Bui N.H., Meens J., Dmytryshyn J., Ho V.W., Tang K.H., Paterson J., Clarke B.A., Bernardini M.Q., Bader G.D., Neel B.G, & Ailles L.E. (2020). Distinct fibroblast functional states drive clinical outcomes in ovarian cancer and are regulated by TCF21. The Journal of Experimental Medicine, 217(8), e20191094.

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Monitoring eIF2α Phosphorylation in HeLa Cells

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HeLa cells were seeded in a 12-well cluster (10⁵ cells/well) and, after overnight incubation, transfected with the indicated plasmids (500 ng well; 250 ng/plasmid) using Lipofectamine2000 (Invitrogen). At 24 hr post transfection, cells were either left untreated or treated with 500 μM sodium arsenite (NaAsO₂, Riedel-de Haën) diluted in DMEM for 30 min at 37°C. Cells were detached with trypsin, washed once PBS, and fixed in PBS, 3.7% PFA for 10 min. To stain for p-eIF2α, cells were permeabilized in ice-cold MeOH for 10min, washed twice in FACS buffer (PBS + 0.02% Na-azide and 1% BSA) and subsequently incubated with the primary antibody rabbit anti-p-eIF2α (1:100, Abcam) in FACS buffer for 45 min. The samples were washed twice in FACS buffer and incubated in the secondary antibody goat anti-rabbit Alexa 594 (1:100, Invitrogen) in FACS buffer for 45 min. Cells were washed twice with FACS buffer and stored at 4°C. Fluorescence intensity was recorded with a CytoFLEX LX flow cytometer (Beckman Coulter) and the data were analyzed by FlowJo v10 software (BD Biosciences). Samples were gated for live single cell populations and then gated for negative, low (L) and high (H) RFP expressing cells. For bar graphs [H/L] ratios were normalized with the ratio calculated for the relevant wildtype protein set at 100%.

Aloise C., Schipper J.G., van Vliet A., Oymans J., Donselaar T., Hurdiss D.L., de Groot R.J, & van Kuppeveld F.J. (2023). SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b. PLOS Pathogens, 19(8), e1011582.

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Hypoxia and CAIX Immunostaining in Xenograft Tumors

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Frozen xenograft tumors were sectioned (5 μm) and stained for hypoxia (pimonidazole) and CAIX. Sections were fixed using cold acetone, rehydrated in TBS with 0.2% Tween-20 (TBS-T) and pre-incubated with 1% normal goat serum (NGS) before exposing them to the primary antibodies: FITC-conjugated IgG1 mouse monocolonal anti-pimonidazole (1:150, clone 4.3.11.3, HP1-Plus Kit, Bio-connect) and rabbit polyclonal anti-CAIX (1:1.000, Novus biologicals). After washing with TBS-T, incubation with the secondary antibody goat anti-rabbit Alexa 594 (1:500) (Invitrogen) was performed. Sections were mounted using fluorescent mounting medium (DakoCytomation) and digitally scanned using an Olympus BX51WI fluorescence microscope with a Hamamatsu EM-CCD C9100 digital camera, a motorized stage (Ludl Mac 2000) and a 10x objective. Micromanager 1.4 software was used for automated image acquisition. Image stitching was performed by using ImageJ software.

van Gisbergen M.W., Offermans K., Voets A.M., Lieuwes N.G., Biemans R., Hoffmann R.F., Dubois L.J, & Lambin P. (2020). Mitochondrial Dysfunction Inhibits Hypoxia-Induced HIF-1α Stabilization and Expression of Its Downstream Targets. Frontiers in Oncology, 10, 770.

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Subcellular Localization of TMEM30A and ATP8A2

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COS7 cells were cultured in Dulbecco’s modified Eagle’s medium/GlutaMAX (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen) and 100 units/ml penicillin/streptomycin (Invitrogen) under 5% CO₂ at 37 °C. The cells were transiently transfected with HA-tagged TMEM30A or Flag-tagged ATP8A2. After 2 days, the transfected cells were fixed with 4% paraformaldehyde, and the TMEM30A proteins were visualized using rat anti-HA antibody (Roche, Redwood City, CA, USA). The ER marker proteins were labeled with a rabbit anti-Calnexin antibody (Cell Signaling Technology, CA, USA), the Golgi markers were labeled with a rabbit anti-GM130 antibody (BD Biosciences, Mississauga, ON). The early endosomes were labeled with an antibody against the specific marker Rab11a (BD Biosciences, Heidelberg, Germany). The secondary antibodies used included goat anti-rabbit Alexa 488, goat anti-rabbit Alexa 594, goat anti-mouse Alexa 488, goat anti-mouse Alexa 594, donkey anti-rat Alexa 488 and donkey anti-rat Alexa 633 (Invitrogen). The images were captured under a Zeiss LSM 800 confocal scanning microscope.

Yang Y., Sun K., Liu W., Zhang L., Peng K., Zhang S., Li S., Yang M., Jiang Z., Lu F, & Zhu X. (2018). Disruption of Tmem30a results in cerebellar ataxia and degeneration of Purkinje cells. Cell Death & Disease, 9(9), 899.

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Immunohistochemical Staining of Brain Tissue

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Sections of brain tissue were washed in 0.15 M PB and blocked with 3% normal goat serum (GIBCO, Carlsbad, CA). For BrdU, antigen retrieval was performed by incubating free floating tissue sections in 4 N HCl for 20 min at room temperature. Staining of free-floating sections was performed using rabbit anti-P2Y12R (1:3000; Anaspec, Fremont, CA), rat anti-BrdU (1:300; Abcam, Cambridge, UK), and rat anti-CD68 (1:1000; Serotec, Raleigh, NC). Secondary antibodies included goat anti-rabbit Alexa 594 (1:2000; Invitrogen) and goat anti-rat Alexa 488 (1:2000; Invitrogen). To visualize BrdU for Fig. 1B, biotinylated goat anti-rat (1:1000; Vector, Burlingame, CA), followed by avidin-biotin complex kit solution (Elite Vectastain ABC, Vector) and subsequent 3-3’ -diaminobenzidine reaction kits (Vector), were used according to the manufacturer’s protocols. Hemoxylin and eoxin (H and E) were used to stain the tumor sections presented in Fig. 7B.

Belcher E.K., Sweet T.B., Karaahmet B., Dionisio-Santos D.A., Owlett L.D., Leffler K.A., Janelsins M.C., Williams J.P., Olschowka J.A, & O’Banion M.K. (2020). Cranial irradiation acutely and persistently impairs injury-induced microglial proliferation. Brain, Behavior, & Immunity - Health, 4, 100057.

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