Sagittal or coronal 50 μm sections containing optic tract, LGN or SC were quenched with 1% Sodium borohydrate for 5 minutes and blocked for 1 h with 3% normal donkey serum and 0.3% Tween 20 in PBS (PBST). Sections were incubated overnight at 4°C with the following primary antibodies: 1:500 Goat anti-biotin (Pierce), 1:200 mouse anti-MAP2 (Millipore), 1:200 mouse anti-NeuN (Millipore), 1:500 rabbit anti-Alix (Millipore), 1:500 mouse anti-Synaptotagmin (MiIlipore). After three washes in PBST, sections were incubated in secondary antibodies: 1:200 dilution of anti-goat alexa 488 or anti-mouse alexa 564 (Invitrogen) for 1 hour at room temperature in blocking buffer. Sections were mounted in Vectashield mounting medium (Vector Labs) and images were collected using a Spinning disc confocal (Ultraview VOX, Perkin Elmer) or a laser scanning confocal (Olympus FV500) microscope. For or obtaining large fields image of rats and pups brains, imaging was performed with Plan Apo 10X objective with 0.45 NA and Plan Apo 20X objective with 0.75 NAon Nikon Alplus confocal system with Andor iXon EMCCD camera system. Large area composite images were made using Nikon NIS elements software using 15% overlap between individual images. All images were background subtracted and processed using Metamorph image processing software.
Rabbit anti alix
Manufactured by Merck Group
Sourced in United States, Canada
Rabbit anti-Alix is a laboratory reagent used in Western blotting and immunoprecipitation applications to detect the Alix protein. Alix is a protein involved in endosomal sorting and multivesicular body formation.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti map2, by Merck Group (2 mentions)
Alplus confocal system, by Nikon (2 mentions)
Anti goat alexa 488, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa564, by Thermo Fisher Scientific (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
Nis elements software, by Nikon (2 mentions)
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Fv500, by Olympus (2 mentions)
Ultraview vox, by PerkinElmer (2 mentions)
Exoquick kit, by System Biosciences (2 mentions)
Imipramine, by Merck Group (1 mentions)
Sr11302, by Bio-Techne (1 mentions)
Mouse anti coxiv, by Cell Signaling Technology (1 mentions)
Probenecid, by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Siramesine, by Merck Group (1 mentions)
Rabbit anti ki67, by Leica Biosystems (1 mentions)
Rabbit anti p2x7, by Alomone (1 mentions)
Mouse anti flotillin 1, by BD (1 mentions)
Arl67156, by Bio-Techne (1 mentions)
6 protocols using rabbit anti alix
1
Immunohistochemistry of Neural Tissues
2
Immunohistochemistry of Neural Tissues
3
Immunocytochemical analysis of P2X7 receptor
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The following antibodies were used: rabbit anti-P2X7 (Alomone Labs), mouse anti-flotillin-1 (BD Bioscience), and rabbit anti-Ki67 (Novocastra). Phalloidin-488 was used to stain F-actin; the monoclonal antibody against desmoyokin-AHNAK (dA) was a gift of Prof. Jacopo Meldolesi (Università Vita-Salute San Raffaele, Milano, Italy). Rabbit anti-Alix (Millipore), goat anti-CD63 (Biorbyt), mouse anti-COX-IV (Cell Signaling Technology), rabbit anti-cleaved caspase-3 (Cell Signaling Technology), β-actin (Sigma). Oxidized ATP (oxATP), brilliant blue G (BBG), probenecid, imipramine, siramesine and actinomycin D (actD) and ethidium bromide were purchased from Sigma-Aldrich; ARL67156 and SR11302 were from Tocris. 10Panx was from Innovagen and 11R-VIVIT from Calbiochem. WP631 was a gift of Dr. Cinthia Farina (San Raffaele Scientific Institute, Milano, Italy).
4
Protein Immunoblotting Analysis Protocol
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Analyzed proteins were quantified and used for immunoblotting analyses. The equal quantified proteins or equal volumes of immunoprecipitated proteins were analyzed by SDS-PAGE. Separated proteins were transferred to nitrocellulose membranes (PolyScreen, PerkinElmer). Transferred antigens were incubated with the following antibodies: mouse anti-galectin-3, rabbit anti-Alix, mouse anti-HIV-1 p24 (Millipore, Burlington, MA, USA), mouse anti-β-actin (Sigma, St. Louis, MO, USA), or rabbit anti-α-tubulin (Epitomics, Burlingame, CA, USA), and held for 1 h at 37 °C. After three washes with 1x phosphate-buffered saline with 0.1% Tween® detergent (PBST), membranes were incubated with horseradish peroxidase (HRP)-conjugated antibodies (goat anti-mouse IgG, goat anti-human IgG, or goat anti-rabbit IgG) (Amersham Biosciences, Charlemagne, UK) for 1 h at 37 °C. Hybridized protein bands were created with an Immobilon™ Western Enhanced Chemiluminescence (ECL) protein detection system (Millipore, Burlington, MA, USA).
5
Exosomes Isolation from 4T1 Cells
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4T1 cells were plated in 60 mm plates at a density of 1.6 × 106 cells per well. Next day, cells were infected with AdEmpty or AdFAST at an MOI of 1000 for 1 h in 500 μl. Following the 1 h infection, 5% FBS/MEM was added to a final volume of 3 ml. Media was isolated 72 h post infection, and exosomes were isolated using the Exoquick kit (System Biosciences, Mountain View, CA, USA) according to the manufacturer's instructions. Purified exosomes were analyzes by immunoblot using rabbit anti-Alix (Sigma, Oakville, Ontario, Canada) at a dilution of 1:1000, rabbit anti-Flotillin-2 (Cell Signaling, Danvers, MA, USA) at 1:1000, or HA at 1:1000. The concentration of exosome particles isolated from media was quantified using Zetaview (Particlemetrix, Germany) according to the manufacturer's instructions. Exosomes were isolated from 3–4 independent experiments, with 11 individual readings for particle size determination per sample for each experiment.
6
Extracellular Vesicle Protein Profiling
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For EV marker protein analysis, the small EVs isolated from equal volumes of plasma of volunteers and rats were subjected to Western blot analysis which was performed according to the standard protocol as previously described.30 The following antibodies were used: rabbit anti‐HSC70 (Proteintech; 10645‐1‐AP; 1:3000), rabbit anti‐Alix (Sigma; ab186429; 1:5000), rabbit anti‐TSG101 (Proteintech; 28283‐1‐AP; 1:2000), rabbit anti‐CD63 (Sigma; ab216230; 1:1000), mouse anti‐Integrin αIIb (SC‐59923; Santa Cruz, 1:1000). For HMGB1 protein analysis, equal amounts of EV proteins (20 μg) were loaded and rabbit anti‐HMGB1 (Proteintech; 10829‐1‐AP; 1:2000) antibody was used.
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