The AR antibody (Catalog: SC-816-G) was from Santa Cruz Biotechnology (Paso Robles, US). Testosterone and DHT were obtained from Sigma (Poole, UK). The SMA antibody (Catalog: A2547) was from sigma. DAPI, Alexa Fluor@488 donkey-anti goat antibody (Catalog: A-11055) and Alexa Fluor@594 goat-anti mouse antibody (Catalog: A-11005) were from Invitrogen (Paisley, UK).
Alexa fluor 488 donkey anti goat antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor@488 donkey-anti goat antibody is a fluorescently labeled secondary antibody used for detection and visualization in various immunoassays and imaging applications. It is designed to bind to primary antibodies raised in goats, and the Alexa Fluor@488 dye provides a green fluorescent signal upon excitation.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 goat anti mouse antibody, by Thermo Fisher Scientific (2 mentions)
Ar antibody, by Santa Cruz Biotechnology (1 mentions)
Testosterone, by Merck Group (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Alexa fluor 488 donkey anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Dm il led optical microscope, by Leica (1 mentions)
Alexa fluor 595 donkey anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Af6000 modular microscope, by Leica (1 mentions)
Mouse monoclonal anti cb2 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti sma, by Merck Group (1 mentions)
Prolong gold anti fade reagent contained dapi, by Thermo Fisher Scientific (1 mentions)
Anti ar antibody, by Santa Cruz Biotechnology (1 mentions)
Fluorescence microscope, by Leica (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Latex beads, by Thermo Fisher Scientific (1 mentions)
Ab 108 c, by R&D Systems (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
9 protocols using alexa fluor 488 donkey anti goat antibody
1
Immunofluorescence Analysis of Androgen Receptor
2
Matrigel-based Angiogenesis Assay in Mice
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We used 6-8 week old wild-type mice from the Jackson Laboratory. Mice (n = 5) were subcutaneously injected with 0.5 mL Matrigel (BD Biosciences) containing 20 µg of SW480-derived EVs or phosphate buffered saline (Invitrogen). On day 7 after injection, the mice were killed, and the Matrigel was removed and stained for whole-mount immunofluorescence. Blood vessels were immunostained with goat anti-CD31 antibody (Cell Signaling Technology) followed by AlexaFluor488 donkey anti-goat antibody (Invitrogen). All images were visualized using an FV1000 Olympus confocal microscope (Olympus, Tokyo, Japan) equipped with a UPlanSApo 20×/0.75 objective lens and acquired using FV1000-ASW 1.5 software (Olympus). The CD31-positive area (µm2) in the immunofluorescence image was quantitatively analyzed using ImageJ software (National Institutes of Health). All animals received humane care, and the experiments were approved by the Institutional Animal Care and Use Committee at Pohang University of Science and Technology, Pohang, Republic of Korea (approval number: 2011-01-0015).
3
Immunofluorescence Visualization of Vimentin, CB2, and PI-PLC β2
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Vimentin, CB2, and PI-PLC β2 were visualized by immunofluorescence. Cells were plated at a density of 8 × 103/cm2 and cultured for 48 h and then, washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C, and permeabilized with 0.5% Triton-X 100 in PBS for 10 min at room temperature. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature, cells were incubated at 1 h, at room temperature, with mouse monoclonal anti-vimentin antibody (Proteintech Group, Manchester, UK) 1:50, mouse monoclonal anti-CB2 antibody 1:150, and mouse monoclonal anti-PI-PLC β2 antibody (Santa Cruz Biotechnology) 1:50. Cells were washed with PBS and then, incubated for 1 h, at room temperature, with Alexa Fluor 488 donkey anti-rabbit antibody 1:300 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), to stain vimentin green; with Alexa Fluor 488 donkey anti-goat antibody 1:600 (Invitrogen, Thermo Fisher Scientific), to stain CB2 receptors green; and Alexa Fluor 595 donkey anti-rabbit antibody 1:300 (Invitrogen, Thermo Fisher Scientific), to stain PI-PLC β2 red. Slides were washed and then, stained with DAPI (Invitrogen, Thermo Fisher Scientific) to visualize the nuclei. The images were captured by a Leica DM IL LED optical microscope, using an AF6000 modular microscope (Leica Microsystem, Milan, Italy).
4
Testosterone Regulation of Androgen Receptor in VSMCs
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VSMCs were seeded on glass coverslips in 12-well plates at a density of 50, 000 cells/well. Following confluence, VSMCs were serum-restricted for 24 hrs and treated with contro or 100 nM testosterone for 48 hrs. Cells were fixed with 4% paraformaldehyde and washed with PBS. The fixed cells were permeabilised with 0.3% Triton-X 100 (Sigma) and incubated with anti-AR antibody (Santa Cruz Biotechnology) and anti-SMA (Sigma) overnight at 4o C. After washing, cells were incubated with Alexa Fluor@488 donkey-anti goat antibody and Alexa Fluor@594 goat-anti mouse antibody (Invitrogen) for 1 hr in the dark. Glass coverslips were mounted onto slides with Prolong®Gold Anti-Fade Reagent contained DAPI (Invitrogen). Fluorescence signal was detected under a Leica fluorescence microscope (Milton Keynes, UK).
5
Flow Cytometry Analysis of Extracellular Vesicles
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Flow cytometry was performed on EVs bound to latex beads based on published protocol66 . Briefly, EVs (3E + 9p) were incubated with 10 μl latex beads (aldehyde/sulfate, 4% w/v, 4 μm, ThermoFisher) and continuously rotated overnight at 4 °C. Glycine (100 mM) was added and beads were further incubated for 30 min. Beads were washed three times with PBS/0.5% BSA (centrifugation 3 min, 4000 rpm) and blocked with 100 μl PBS/2.5%BSA for 30 min. Beads were incubated for 30 min on ice with primary goat FGF2 antibody (AF233, R&D Systems) or goat IgG control (AB108C, R&D Systems). Beads were washed with PBS and incubated in 100 μl for 30 min on ice with secondary Alexa Fluor 488 donkey anti-goat antibody (1:500, Invitrogen, ThermoFisher). After wash, beads were resuspended in 800 μl and analyzed on a FC500 (Beckman coulter) flow cytometer with CXP software.
6
Spinal Cord Immunohistochemistry Protocol for NOS-II and Iba-1
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Rats were deeply anesthetized with 3% isoflurane in a mixture of N2O/O2 gas at day 5 post-CCI surgery and perfused transcardially with calcium-free Tyrode’s solution and subsequently with fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The spinal cords were collected after perfusion, postfixed in the identical fixative for 2 h at RT and then placed in 30% sucrose in PBS (pH 7.4) at 4°C. Serial transverse sections (40 μm) of the L4–5 spinal cord were cut using a cryostat (Leica CM1520, Leica Biosystems, Germany). Spinal tissue sections were washed with PBS and incubated for 2 days at 4°C with a primary antibody specific for NOS-II (rabbit polyclonal anti-NOS2 antibody, 1:1,000, cat# sc-651, Santa Cruz Biotechnology Inc.) or Iba-1 (goat polyclonal anti-Iba-1 antibody, 1:500, cat# ab5076, Abcam plc.). The primary antibodies were detected by incubating the tissue in Alexa Fluor® 568 donkey anti-rabbit antibody (1:400, Invitrogen) or Alexa Fluor® 488 donkey anti-goat antibody (1:400, Life Technologies) for 90 min at RT. Tissue sections were mounted on slides and visualized with a confocal microscope (Nikon Eclipse TE2000-E, Nikon, Japan).
7
Semaphorin-7A Immunofluorescence Staining
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HLF were treated with either control or semaphorin-7A siRNA and cultured for 24h. Cells were subsequently plated into 8-well chamber-slides (Ibidi, Munich, Germany) at 1.65x105 per ml and allowed to adhere for 24 h. The cells were washed twice with TBS, fixed with 4% paraformaldehyde/TBS for 30 min at room temperature (RT) and permeabilized with 0.2% Triton X-100/TBS (Thermo Fisher Scientific, Rockford, IL) for 5 min at RT. Cells were then blocked with 10% BSA in TBS for 1h at RT and incubated overnight at 4°C with the relevant primary antibody (1.3μg/ml in 2% BSA/0.2% Triton X-100/TBS). Cells were washed with TBS, incubated with secondary antibody in 2% BSA/TBS for 75 min at 37°C, washed with TBS, incubated with DAPI/TBS (0.5μg/ml) for 10 min RT, washed with TBS and mounted in Ibidi mounting media (Munich, Germany). Goat polyclonal anti—semaphorin-7A Ab and goat IgG control were from R&D Systems (Minneapolis, MN). Secondary Alexa Fluor-488 donkey anti goat antibody was from Life Technologies (Eugene, OR). Immunofluorescent images were obtained using an Olympus 1X71 fluorescent microscope and Q imaging Retiga 2000R camera.
8
Immunofluorescent Localization of GRP78/BiP
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After culture, the cells were washed and resuspended in PBS and then transferred onto poly-L-lysine glass slides (Menzel-Glaser, Braunschweig, Germany). The bound cells were washed twice in PBS, fixed with cooled methanol (−20 °C) for 20 min, washed in PBS, and then blocked with 0.2% bovine serum albumin (BSA) in PBS for 1 h at 37 °C. The cells were then incubated with primary antibody against GRP78/BiP diluted 1:1000 for 1 h at 37 °C in PBS containing 0.2% BSA, washed twice in PBS containing 0.2% BSA, and then incubated with Alexa Fluor 488 donkey anti-goat antibody (Life Technologies Corporation, Eugene, OR, USA) in PBS containing 0.2% BSA for 1 h at 37 °C. The specimens were washed twice in PBS containing 0.2% BSA and were labeled with 10 mg/L 4’-6-diamidino-2-phenylindole (DAPI, Sigma Aldrich, St. Louis, MO, USA) in PBS to visualize the nuclei. Finally, the coverslips were mounted onto slides with a mounting medium (80% glycerol) and analyzed using a confocal laser scanning microscope (Nikon Eclipse Ti, Tokyo, Japan).
9
Visualizing Intestinal Tight Junctions
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