Total protein was extracted from cerebral cortex of brain hemisphere. Briefly, brain tissue was homogenized in RIPA lysis buffer (CST) with further centrifugation at 14,000× g at 4°C for 30 min. Protein concentration in the supernatant was determined using the BCA assay kit (Bio-Rad). Equal amounts of protein were loaded on a SDS–PAGE gel. After being electrophoresed and transferred to a PVDF membrane, the membrane was blocked and incubated with the primary antibodies against Synapsin 1 (CST; 1:2,000), Post-synaptic density protein 95 (PSD95; Thermo Fisher Scientific, Waltham, MA, USA; 1:2,000), Aβ (CST; 1:2,000), actin (Sigma; 1:6,000), or GAPDH (Abcam; 1:5,000) overnight at 4°C. The membrane was then incubated with fluorescence-labeled secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature against light. After washing, the membrane was then scanned with the Odyssey CLx Imaging System. The data were analyzed with the NIH ImageJ software. The values in the figures represent relative density of the bands normalized to GAPDH/actin.
Fluorescence labeled secondary antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Fluorescence-labeled secondary antibodies are laboratory reagents used in various immunochemical techniques. They bind to primary antibodies and emit fluorescent signals, allowing for the detection and visualization of target molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Zen 2008, by Zeiss (3 mentions)
Lsm710 confocor3 lsm, by Zeiss (3 mentions)
Hoechst 33342, by Merck Group (3 mentions)
Axio observer z1, by Zeiss (3 mentions)
Nis elements advanced research software, by Nikon (3 mentions)
Eclipse te2000 u inverted microscope, by Nikon (3 mentions)
Prolong gold antifade reagent with dapi, by Cell Signaling Technology (3 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Merck Group (2 mentions)
Secondary horseradish peroxidase hrp conjugated antibodies for less, by Merck Group (2 mentions)
Odyssey scanner, by LI COR (2 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Image reader las 4000, by Fujifilm (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
Prolong diamond antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Nile red, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Paraformaldehyde (pfa), by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
36 protocols using fluorescence labeled secondary antibody
1
Quantifying Synaptic Proteins in Brain
2
Immunofluorescence Staining of Pancreatic Cell Markers
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Immunofluorescence staining was performed as previously described.17 (link) Briefly, at room temperature, cells were fixed with 4% formaldehyde for 30 min and then treated with 0.25% Triton X-100 in PBS for 10 min. After being blocked with 10% normal donkey serum containing 1% BSA in PBS, cells were incubated with primary antibodies against Ins2 (1:200; Cell Signaling Technology), NeuroD1 (1:200; Santa Cruz), or Pdx1 (1:1,000; Abcam) at 4°C overnight. Fluorescence-labeled secondary antibodies were used for detection (Thermo Scientific). Nuclei were stained with DAPI (1 mg/mL; Roche). Images were acquired via fluorescence microscopy.
3
Protein Detection by Immunoblotting
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Immunoblotting was performed using a standard protocol. Proteins were separated in NuPAGE (4–12%) Bis–Tris gels (Thermo Fisher) and transferred onto nitrocellulose membranes (Bio-Rad). The target protein was detected by specific primary antibodies followed by secondary horseradish peroxidase (HRP)-conjugated antibodies (for less abundant proteins) (Sigma) or fluorescence-labeled secondary antibodies (for abundant antigens) (Thermo Fisher). For immunoblotting with HRP-conjugated secondary antibodies, the signal was detected by the enhanced chemiluminescence method (ECL) using the Immobilon western chemiluminescent HRP substrate (Millipore), and recorded by a Fuji LAS-4000 image reader. The intensity of the detected protein bands was quantified by ImageGauge v3.0 software. For immunoblotting with fluorescence-labeled secondary antibodies, membranes were scanned using a LI-COR Odyssey scanner. The intensity of the protein bands was quantified by the LI-COR Odyssey v3.1 software.
4
Antibody Landscape for Cellular Analysis
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The following antibodies were purchased: rabbit polyclonal anti-Rnd2 from Proteintech (Rosemont, IL); mouse monoclonal anti-myelin basic protein (MBP), and rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) from Covance (Princeton, NJ); mouse monoclonal anti–β-actin from Fujifilm (Tokyo, Japan); goat polyclonal anti–platelet-derived growth factor receptor (PDGFR) α from Santa Cruz Biotechnology (Santa Cruz, CA); mouse monoclonal anti-MBP, mouse monoclonal anti–adenomatus polyposis coli (APC) (also called CC1), mouse monoclonal anti-O4, and mouse monoclonal anti-NeuN from Merck-Millipore (Billerica, MA); rabbit polyclonal anti–phosphorylated Thr696-Mbs and rabbit polyclonal anti-Mbs from Cell Signaling Technology (Danvers, MA); rabbit monoclonal anti-Olig2, rabbit polyclonal anti–phosphorylated Ser1366-Rho kinase, which recognizes active states, and rabbit polyclonal anti-Rho kinase from Abcam (Cambridge, UK); mouse monoclonal anti-2′, 3′-cyclicnucleotide 3′-phosphodiesterase (CNPase) and rabbit polyclonal anti-neurofilament (NF) large subunit from Sigma-Aldrich (St. Louis, MO); peroxidase-conjugated secondary antibodies from MBL (Nagoya, Japan); and fluorescence-labeled secondary antibodies from Thermo Fisher Scientific (Waltham, MA).
5
Quantifying Sclerostin and MyoD in C2C12 and C57 Cells
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C2C12 and C57 cells grown on glass coverslips were fixed in 4% paraformaldehyde at 4 °C for 20 min, blocked in 4% (w/v) bovine serum albumin (BSA) PBS and incubated with primary anti-sclerostin (ab63097, Abcam, Cambridge, United Kingdom) anti-MyoD antibody (Santa Cruz Biotechnology, Dallas, USA) overnight at 4 °C. Coverslips were next washed with PBS and incubated with fluorescence-labeled secondary antibodies (Thermo Scientific, Waltham, MA, USA) for 1 h at room temperature. Slides were mounted with a 10% DABCO (1,4-diazabicyclo[2.2.2]octane) solution and were observed using a Nikon A1 confocal laser scanning microscope. The confocal serial sections were processed with ImageJ software to obtain three-dimensional projections and image rendering was performed using Adobe Photoshop CS 8.0 software (Adobe Systems, San Jose, CA, USA). All the images shown in this paper are representative of at least 3 independent experiments carried out under the same conditions.
6
Immunofluorescence protocol for paraffin and OCT sections
7
Comprehensive Antibody Sourcing and Validation
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The following antibodies were purchased: anti-Rab35 from the Proteintech Group (Chicago, IL); anti-MBP from Covance (Princeton, NJ) or Merck Millipore (Billerica, MA); anti–β-actin from BD Biosciences PharMingen (Franklin Lakes, NJ); anti-Arf6 and anti–cytohesin-2 from Santa Cruz Biotechnology (Santa Cruz, CA); anti-ACAP2 from Abcam (Cambridge, UK); anti–green fluorescent protein (anti-GFP) from MBL (Nagoya, Japan); anti-ZsGreen from Clontech Takara Bio (Kyoto, Japan); anti-FLAG from Sigma-Aldrich (St. Louis, MO); anti-myc from Nacalai Tesque (Kyoto, Japan) or MBL; horseradish peroxidase–conjugated anti-mouse, anti-rabbit, or anti-goat immunoglobulin G secondary antibodies from GE Healthcare (Little Chalfont, Buckinghamshire, UK); and fluorescence-labeled secondary antibodies from Thermo Fisher Scientific (Waltham, MA).
8
Quantitative Immunoblot Analysis Protocol
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Immunoblotting was performed using a standard protocol. Proteins were separated in NuPAGE (4%-12%) Bis-Tris gels (Thermo Fisher) and transferred onto nitrocellulose membranes (Bio-Rad). The target protein was detected by specific primary antibodies followed by secondary horseradish peroxidase (HRP)-conjugated antibodies (for less abundant proteins) (Sigma) or fluorescence-labeled secondary antibodies (for abundant antigens) (Thermo Fisher). For immunoblotting with HRP-conjugated secondary antibodies, the signal was detected by the enhanced chemiluminescence method (ECL) using the Immobilon western chemiluminescent HRP substrate (Millipore) and recorded by a Fuji LAS-4000 image reader. The intensity of the detected protein bands was quantified by ImageGauge v3.0 software. For immunoblotting with fluorescence-labeled secondary antibodies, membranes were scanned using a LI-COR Odyssey scanner. The intensity of the protein bands was quantified by the LI-COR Odyssey v3.1 software.
9
Immunofluorescence Staining of Cells
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Cells were seeded on coverslips coated with gelatin at an appropriate density. Cells were fixed by 4% paraformaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature and blocked with the blocking buffer (1X PBS, 0.2% Triton, 3% BSA) for 30 min. Cells were then incubated with the primary antibody for 2-3 h at room temperature, washed three times with 0.2% Triton X-100 in PBS, then incubated with fluorescence-labeled secondary antibodies (Invitrogen, 1:500 dilution) for 30 min at room temperature and rewashed three times. A drop (10 μL) of VECTASHIELD antifade mounting medium containing 4′,6′-diamidino-2-phenylindole (DAPI) (Vector labs, H-1200) was added on a labeled microscope slide. The coverslip was then placed on the drop, observed and photographed under the Carl Zeiss LSM 710 confocal fluorescence microscope.
10
Immunofluorescence Analysis of hERG and α-Actinin
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The details of the experiments protocol were performed as described previously [15 (link)]. Primary antibodies against hERG (1:50, Santa Cruz) and α-actinin (1:200, Cell Signaling, Beverly, MA, USA) were applied following by incubation with the appropriate fluorescence-labeled secondary antibodies (Invitrogen) for 1 h at room temperature. After several washes, the samples were air-dried, mounted with a drop of ProLong Diamond Antifade Mountant with DAPI (Molecular Probes) and subjected to microscopy. Images were acquired using a 100× oil objective (Plan-Apochromat 100× [numerical aperture, 1.46] oil immersion objective for differential interference contrast [DIC]; Carl Zeiss). All sections were analyzed using a confocal laser microscopy system and software (LSM710; Carl Zeiss) that was built around an inverted microscope (Axio Observer Z1; Carl Zeiss, Germany). Images were saved in TIFF format and analyzed by ImageJ software (Wayne Rasband, National Institutes of Health, USA).
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