Alexa 594 conjugated goat anti rabbit secondary antibody

Manufactured by Thermo Fisher Scientific

Sourced in United States

Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody is a fluorescently labeled reagent used to detect and visualize rabbit primary antibodies in various immunoassay applications. It provides a bright, stable, and specific signal for the detection of target proteins or antigens.

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Lab products found in correlation

Axio imager, by Zeiss (4 mentions) Mounting medium with dapi, by Zeiss (2 mentions) Triton x 100, by Merck Group (2 mentions) β catenin, by Abcam (1 mentions) Click it plus tunel assay for in situ apoptosis detection, by Thermo Fisher Scientific (1 mentions) Piezo1, by Novus Biologicals (1 mentions) Yap taz, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Goat serum, by Merck Group (1 mentions) E cadherin, by Abcam (1 mentions) Aggrecan, by Abcam (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Col2a1, by Abcam (1 mentions) Col10, by Abcam (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions) P phenylenediamine, by Merck Group (1 mentions) Fluoview f1000 confocal microscope, by Olympus (1 mentions) Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions) Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions) Bx43 microscope, by Olympus (1 mentions)

10 protocols using alexa 594 conjugated goat anti rabbit secondary antibody

Somatostatin Interneuron Identification

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Under urethane anesthesia, animals (n=3) were transcardially perfused with 4% paraformaldehyde in PBS, and the brain rapidly extracted. Coronal sections (50 μm) were cut, blocked in a solution of 4% normal goat serum (NGS) and incubated overnight with an anti-somatostatin antibody previously validated by indirect immunofluorescence labeling of hypothalamic sections (Immunostar, rabbit) at a dilution of 1:500. Slices were subsequently washed in PBS, incubated for 2 hr with an Alexa-594 conjugated goat anti-rabbit secondary antibody (Life Technologies) at a concentration of 1:500, and washed a second time. After mounting (Vectashield mounting medium with DAPI) and imaging (Zeiss Axio Imager), Arch-expressing somata were first identified, blind to somatostatin immunoreactivity, and thereafter scored for colocalization.
In separate immunohistochemical experiments we detected negligible overlap between cre-expressing somatostatin cells and cells expressing parvalbumin (data not shown). Although we cannot exclude the possibility that some SOM-cre neurons express markers of other interneuron subtypes, the most parsimonious explanation for our results using SOM-cre mice is that we are predominantly manipulating SOM cells.

Sturgill J.F, & Isaacson J.S. (2015). Somatostatin-expressing interneurons provide subtractive inhibition and regulate sensory response fidelity in olfactory cortex. Nature neuroscience, 18(4), 531-535.

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Somatostatin Interneuron Identification

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Comprehensive Immunofluorescence Labeling Protocol

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Samples were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. Fixative was removed by washing three times for 5–10 min in PBS, followed by washing three times in 0.3% Triton X-100 (Sigma Aldrich) in PBS (PBST). Samples were blocked in 10% (vol/vol) goat serum (Sigma Aldrich) in PBST for 1 h, then incubated overnight at 4°C with primary antibodies to label E-cadherin (Abcam), beta-catenin (Abcam), Ki67 (Abcam), Yap/Taz (Cell Signaling), Cx43 (Cell Signaling), cleaved caspase-3 (Cell Signaling), or Piezo1 (Novus). Following incubation with primary antibody, samples were washed three times in PBST, then incubated with Alexa 594–conjugated goat anti-rabbit secondary antibody (Invitrogen) overnight at 4°C. Prior to imaging, samples were washed three times in PBS. Nuclei were visualized by incubating the cells for 15 min in Hoechst 33342 diluted 1:1000 in PBS, followed by three washes with PBS. To visualize proliferation, we used the Click-iT EdU Alexa Fluor 488 or 594 Imaging Kits (Thermo Fisher Scientific). Tissues were exposed to EdU-containing growth medium for 30 min prior to fixation and detection. To detect DNA damage, we used the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection (Thermo Fisher Scientific).

Silver B.B., Wolf A.E., Lee J., Pang M.F, & Nelson C.M. (2020). Epithelial tissue geometry directs emergence of bioelectric field and pattern of proliferation. Molecular Biology of the Cell, 31(16), 1691-1702.

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Immunohistochemical Analysis of mAKAP in Mouse Retina

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2-month old mice were euthanized by 100% CO₂ inhalation. Eyes were dissected and embedded in OCT for cryosection (10 μm) immediately. Sections were post-fixed in 4% PFA for 15 min and then washed 3 times in PBS. Retinal sections were blocked in 5% normal goat serum and 0.2% BSA in PBS for 30 min, then incubated for 1 h in the same buffer with FL100 rabbit antibody to mAKAP 245–340 (Li et al., 2013 (link)). After washing, retinal sections were incubated with Alexa 594-conjugated goat anti–rabbit secondary antibody (1:500; Invitrogen) for 1 h, before washing and mounting.

Wang Y., Cameron E.G., Li J., Stiles T.L., Kritzer M.D., Lodhavia R., Hertz J., Nguyen T., Kapiloff M.S, & Goldberg J.L. (2015). Muscle A-Kinase Anchoring Protein-α is an Injury-Specific Signaling Scaffold Required for Neurotrophic- and Cyclic Adenosine Monophosphate-Mediated Survival. EBioMedicine, 2(12), 1880-1887.

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Immunostaining of Cells and 3D Cultures

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For immunostaining of cells in monolayers, cells were fixed in 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 10 min and then permeabilized with cold 0.4% Triton X-100 (Sigma) in PBS for 15 min. The cells were then incubated for 1 hour in PBS containing 1% BSA–10% FBS–0.4% Triton X-100 (for blocking). For 3D culture staining, hydrogels were fixed in 4% paraformaldehyde overnight, transferred to 70% ethanol, and included in paraffin (Histoserv Inc.). Sections were then deparaffinized through xylene and graded alcohols. A step of enzymatic antigen retrieval with 0.1% Trypsin in PBS was performed prior to blocking (2% goat serum–3% BSA–0.1% Triton X-100) for 1 hour.
After the blocking step, antibodies against Aggrecan (1:500), Col2a1 (1:100; Abcam), Col10 (1:100; Abcam), Sox9 (1:100; Santa Cruz), or anti-human Ki67 (1:500; ebiosciences) were added overnight in blocking buffer. The following day, cells were washed three times with PBS and then incubated for 1 hour at room temperature with Alexa 594-conjugated goat anti-rabbit secondary antibody (1:250; Gibco, Invitrogen). Cellular DNA was counterstained with DAPI (Life Technologies, Carlsbad, CA, USA). The antibody anti-AGC used for this study was a kind gift from Prof. R.L. Smith [23 (link)].

Lee J., Smeriglio P., Chu C.R, & Bhutani N. (2017). Human iPSC-derived chondrocytes mimic juvenile chondrocyte function for the dual advantage of increased proliferation and resistance to IL-1β. Stem Cell Research & Therapy, 8, 244.

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Immunohistochemical Analysis of Basigin and MCT1 in Murine Pineal and Retinal Tissues

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Mouse pineal glands and neural retinas were fixed with 4% paraformaldehyde in 0.1 M cacodylate (pH 7.4) and embedded in paraffin wax as previously described (Ochrietor et al., 2001 (link)). Tissues were sectioned at 6 µm, applied to gelatin-coated slides, deparaffinized, and incubated in blocking solution (TBS containing 0.1% Tween 20 and 2% normal goat serum [Pierce/Thermo Scientific]) overnight at 4°C. The tissues were incubated with antibodies specific for Basigin gene products (Ochrietor et al., 2003 (link)) or MCT1 (Millipore Corporation, Billerica, MA), diluted to 1 µg/mL in blocking solution, for 1 hour at 37°C and then at 4°C overnight. After washing with several changes of TBS, the tissues were incubated with Alexa 594-conjugated goat-anti-rabbit secondary antibody (Invitrogen Corporation), diluted 1:1000 in blocking solution, for 1 hour at 37°C. Coverslips were mounted with 30% glycerol containing p-phenylenediamine (Sigma Chemical Company, St. Louis, MO) and the tissues were viewed with an Olympus Fluoview F1000 confocal microscope (Pittsburgh, PA). Images were gathered digitally using Olympus FV10-ASW 4.0 software and assembled for publication using Microsoft PowerPoint software.

Tokar D., van Ekeris L., Linser P.J, & Ochrietor J.D. (2016). CHARACTERIZATION OF THE EXPRESSION OF BASIGIN GENE PRODUCTS WITHIN THE PINEAL GLAND OF MICE. Cellular and molecular neurobiology, 37(6), 1141-1145.

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Immunofluorescence Staining of E-cadherin in A549 Cells

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A549 cells cultured on 8-well chamber slides were fixed with ice cold methanol for 10 min at −20 °C freezer. Fixed cells were then incubated with phosphate-buffered saline (PBS) containing 0.02 % Tween 20 (PBST) with 10 % goat serum and 5 % BSA for 1 h followed by anti-E-cad antibodies (1:200) in PBST containing 1 % BSA for 1 h at room temperature. After three washes with PBS, the cells were incubated with Alexa 594-conjugated goat anti-rabbit secondary antibody (1:1000, Invitrogen) for 1 h, which was followed by another three washes with PBS. Cells were then stained with DAPI for 10 min at room temperature. After washing by PBS, the chambers were removed and the slides were mounted with ProLong Gold Antifade Mountant (Life Technologies, Grand Island, NY) and covered with cover slips. Cells were examined with an Olympus BX43 microscope (OLYMPUS Corporation, Tokyo, Japan).

Dong C., Gongora R., Sosulski M.L., Luo F, & Sanchez C.G. (2016). Regulation of transforming growth factor-beta1 (TGF-β1)-induced pro-fibrotic activities by circadian clock gene BMAL1. Respiratory Research, 17, 4.

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Quantifying Smad2/3 Translocation in Lung Fibroblasts

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Primary human lung fibroblasts (HLFs) were seeded in 96-well tissue culture plates pre-coated with a 0.2 % gelatin solution. After 48 h, culture media was replaced with serum-free DMEM media containing 5uM hit compounds. Cells were pretreated with compounds for 30 min at 37 ℃ before TGF-β1 was added to a final concentration of 2.5 ng/ml. Two hours after TGF-β1 addition, cells were processed for Smad2/3 staining. Briefly, cells were washed with PBS, fixed with 4 % formaldehyde for 15 min, and permeabilized with 0.2 % Triton for 10 min. After blocking with 3 % BSA for 1hr, cells were incubated with rabbit anti-Smad2/3 antibody (Cell Signaling Technology, Cat #8685, diluted 1000-fold in 1 % BSA) for 1hr, washed, and stained with an Alexa 594-conjugated Goat anti-rabbit secondary antibody (Invitrogen). Nuclei were counterstained with Hoechst. Cells were imaged on an ImageXpress Confocal microscope (Molecular Devices) using a 20x objective. The cytoplasmic/ nuclear intensity ratio was quantified using CellProfiler [22] . Each hit was measured in triplicate.

Wang Y., Cortes E., Huang R., Wan J., Zhao J., Hinz B., Damoiseaux R, & Pushkarsky I. (2024). FLECS technology for high-throughput screening of hypercontractile cellular phenotypes in fibrosis: A function-first approach to anti-fibrotic drug discovery. SLAS discovery : advancing life sciences R & D, 29(3).

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Epithelial-Mesenchymal Transition Markers

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QuantiTect primer assays for α-SMA, vimentin, S100A4, Snail, Slug, Twist and glyceraldehyde 3-phosphate dehydrogenase were from Qiagen (Valencia, CA, USA). E-cadherin monoclonal antibody for immunofluorescence studies), β-catenin polyclonal antibodies (for immunofluorescence studies) and α-SMA polyclonal antibodies were from AbCam (Cambridge, MA, USA). Alexa 488-conjugated goat anti-mouse and Alexa 594-conjugated goat anti-rabbit secondary antibodies were from Life Technologies (Carlsbad, CA, USA). E-cadherin monoclonal antibody (for immunoblotting studies), β-catenin monoclonal antibody (for immunoblotting studies) and horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were from Cell Signaling Technologies (Danvers, MA, USA). Horseradish peroxidase-linked goat anti-mouse secondary antibodies were from GE Healthcare (Pittsburgh, PA, USA). Col IV was from BD Biosciences (San Jose, CA, USA). Uncoated and Matrigel-coated Transwell plates and heparin sodium salt were from Fisher Scientific (Waltham, MA, USA).

Franzen C.A., Blackwell R.H., Todorovic V., Greco K.A., Foreman K.E., Flanigan R.C., Kuo P.C, & Gupta G.N. (2015). Urothelial cells undergo epithelial-to-mesenchymal transition after exposure to muscle invasive bladder cancer exosomes. Oncogenesis, 4(8), e163-.

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Quantifying Activated Microglia in Monkey Cortex

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For detection of activated microglia within the monkey cortex, IBA-1 immunohistochemistry was performed. Forty-micron transverse sections through the monkey motor cortex were blocked with normal goat serum in TBS + 0.25% triton-x. Sections were then incubated overnight at 4C in blocking solution containing rabbit anti-IBA-1 (Wako; 1 μg/ml). Following washes in TBS, sections were incubated for 2 h at room temperature in blocking solution containing Alexa-594 conjugated goat anti-rabbit secondary antibodies (Life Technologies; 1:500). Sections were washed in TBS, mounted on gelatin-subbed slides and coverslipped with Flouromount G. Images from 4 sections per subject were obtained using an Olympus FV1000 laser scanning confocal microscope with a 60 × objective. Percent area occupied by IBA-1 immunolabeling was determined using Image J. Mean density per subject was calculated for each cortex.

Brock J.H., Rosenzweig E.S., Yang H, & Tuszynski M.H. (2017). Enhanced axonal transport: A novel form of “plasticity” after primate and rodent spinal cord injury. Experimental neurology, 301(Pt A), 59-69.

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