Human iPSCs were fixed in 4% PFA at room temperature and then stained with antibodies against the following makers: Oct3/4 (1:500 dilution Abcam #ab27985) and SSEA-4 (1:100 dilution Abcam# ab16287) and AlexFluor-488 conjugated antibodies against human TRA-1-60 (1:100 dilution BD Pharmigen #560173) in 0.1% Triton (Fisher X-100) and and 1% Fetal Bovine Serum (FBS) in PBS. For secondary antibodies goat IgG (1:250 in PBS, Invitrogen #A11055) and mouse IgG (1:250 in PBS, Invitrogen #A11055) were used to detect Oct3/4 and anti-SSEA-4, respectively. Cell nuclei were counterstained with Hoechst 33342 (10 ng/mL of Thermo Scientific #H1399).
A11055
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Japan, France
The A11055 is a laboratory instrument designed for the quantification and analysis of analytes in a variety of sample types. It utilizes advanced spectroscopic techniques to provide accurate and reliable measurements. The core function of the A11055 is to enable researchers and scientists to conduct precise quantitative analyses as part of their research and testing procedures.
Automatically generated - may contain errors
Lab products found in correlation
A21202, by Thermo Fisher Scientific (24 mentions)
A21207, by Thermo Fisher Scientific (21 mentions)
A21206, by Thermo Fisher Scientific (20 mentions)
A31572, by Thermo Fisher Scientific (17 mentions)
A10042, by Thermo Fisher Scientific (12 mentions)
A31571, by Thermo Fisher Scientific (12 mentions)
A21203, by Thermo Fisher Scientific (11 mentions)
Ab6658, by Abcam (11 mentions)
A31573, by Thermo Fisher Scientific (10 mentions)
Ab6673, by Abcam (9 mentions)
A21432, by Thermo Fisher Scientific (8 mentions)
A11058, by Thermo Fisher Scientific (8 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (8 mentions)
A10040, by Thermo Fisher Scientific (7 mentions)
A11008, by Thermo Fisher Scientific (6 mentions)
A11029, by Thermo Fisher Scientific (6 mentions)
A11034, by Thermo Fisher Scientific (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (5 mentions)
A11001, by Thermo Fisher Scientific (5 mentions)
Mab377, by Merck Group (5 mentions)
152 protocols using a11055
1
Pluripotency Marker Expression in hiPSCs
2
Immunohistochemistry of Neuronal Markers
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Immunohistochemistry was performed as described previously37 (link). Briefly, 10 μm thick cryosections were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated in 0.1% Triton X100/phosphate-buffered saline (PBS) for 20 min at 37 °C followed by incubation in 3% bovine serum albumin/PBS for 1 h at room temperature. The cryosections were then incubated with the following primary antibodies for 1 day at 4 °C: rabbit polyclonal antibody against α7-nAChR (sc-5544, 1:20; Santa Cruz Biotechnology), rabbit polyclonal antibody against GAD65/GAD67 (ab11070, 1:200; Abcam), rabbit polyclonal antibody against GABA (A2052, 1:100; Sigma, St. Louis, MO, USA), and goat polyclonal antibody against Brn3a (sc-31984, 1:40; Santa Cruz Biotechnology). The secondary antibodies (all from Invitrogen-Molecular Probes, Eugene, OR, USA) were Alexa Fluor 555-conjugated donkey anti-rabbit IgG antibody (A31572, 1:1,000), Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A11070, 1:500), and Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (A11055, 1:500). The sections were finally counterstained with the nucleic acid stain Hoechst 33258 (H3569, 1:2,000; Invitrogen-Molecular Probes) in PBS and imaged using a laser scanning confocal microscope (TCS SP8; Leica Microsystems, Heidelberg, Germany).
3
Immunofluorescence Staining of Cells
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To fix samples/cells, slides/coverslips were fixed and permeabilised for 15 min in dry methanol at −20 °C and rehydrated in PBS. Samples/cells were blocked (donkey serum 1:30) for 1 h at 37 °C in a humified chamber incubated with primary antibody (either Anti-pSer473AKT1 or TRIB2) for 1 h at 37 °C in a humified chamber followed by PBS washing and incubation with a fluorescent secondary antibody (Molecular Probes A21207 or A11055) for 1 h at 37 °C in a humified chamber. Antibody solutions were made in PBS (Sigma). Following labelling procedures, samples/cells were mounted on glass slides in Clear Mount mounting solution (Invitrogen).
4
Immunofluorescence Staining of Cultured Cells
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Cells, grown on plastic or cover-slips, were fixed in 4% paraformaldehyde (15 min, room temperature), washed three times (phosphate-buffered saline (PBS), 5 min, room temperature), permeabilized (0.5% Triton X-100 in PBS, 5 min, room temperature) and incubated with the primary antibodies diluted 1:20–1:400 (1 h, room temperature or 4°C overnight) in 10% serum in PBS. Cells were then washed three times (PBS, 5 min, room temperature), incubated with secondary antibodies diluted 1:1000 (1 h, room temperature) in 10% serum in PBS, washed again (PBS, 5 min, room temperature), DAPI stained (5 min, room temperature), washed (PBS, 5 min, room temperature) and mounted on a microscope slide with anti-fade (Dako, Glostrup, Denmark). Cells grown on plastic were kept in PBS until imaged. Detection was carried out using the relevant secondary antibodies conjugated to Alexa488 (A21202, A21206, A11055) or Alexa568 (A1003, A10042, A11057, Molecular Probes).
5
Immunochemistry and Immunofluorescence Protocol for Brain Sections and Cultured Neurons
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Immunochemistry and immunofluorescence were performed as described previously.63 (link),64 (link) Frozen 20-μm thick brain sections or cultured neurons placed on Confocal dish (Corning, CLS-DL-CC-014) were fixed in 4% paraformaldehyde, blocked by 8% normal donkey serum (Santa Cruz Biotechnology, sc-2044), and incubated in specific primary antibodies as follows: goat anti-LC3 (1:500), mouse anti-TUBB3 (1:1000), rabbit anti-cleaved CASP3 (1:200), mouse anti-ARRB1 (1:200) and rabbit anti-BECN1 (1:500). After being washed 3 times by PBST (0.1% Tween 20 in PBS [70011-044, Gibco]), the sections and cells were incubated with corresponding Alexa 488-conjugated donkey anti-mouse (Molecular Probes, A-21202), Alexa 555-conjugated donkey anti-rabbit (Molecular Probes, A-21432) and Alexa 647-conjugated donkey anti-goat (Molecular Probes, A11055) secondary antibodies. DAPI (Molecular Probes, D1306) was used to stain nuclei.65 (link) The immunofluorescence TUNEL assay was performed according to the instructions of the manufacturer.8 (link),55 (link) Images were obtained by confocal microscopy (Olympus, Fluoview FV1000, Tokyo, Japan).
6
Immunofluorescence Staining of Cultured Cells
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Cultured cells were fixed with 4% PFA for 30 min at room temperature and then made permeable with 0.5% Triton X-100 in PBS for 15 min. Cells were blocked with 3% BSA in PBS for 30 min and incubated overnight at 4°C with primary antibodies diluted in 1% BSA in PBS. The antibodies used were HNF4A (1:500; Santa Cruz Biotechnology, sc-6556), GATA4 (1:250; Santa Cruz Biotechnology, sc-1237), and NKD1 (1:250; Abcam, ab133650). Cells were washed in 1% BSA in PBS and incubated with secondary antibody (Molecular Probes, A21206, Alexa fluor anti-rabbit 488 nm; A11058, Alexa fluor anti-goat 594 nm; and A11055, Alexa fluor anti-goat 488) and DAPI for 1 h at room temperature and washed with PBS. Images were processed using Adobe Photoshop to optimize brightness and contrast, with all control and experimental images being treated identically.
7
Wholemount Immunofluorescence of Tail Tissues
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Wholemount immunofluorescence staining of tail tissues was performed as previously described (Osorno et al., 2012 (link)). At least two embryos per genotype were stained with each antibody with similar results. Primary antibodies (1:200): goat anti-Brachyury (R and D Systems, AF2085), rabbit anti-Sox2 (Abcam, AB92494), goat anti-Cdh1 (R and D Systems, AF648), rabbit anti-Cdh2 (Abcam, AB18203), goat anti-Tbx6 (R and D Systems, AF4744), rabbit anti-Laminin 111 which detects all laminins containing a1, b1 or g1 chains (Sigma, L9393), goat anti-human EpCAM/TROP1 (R and D Systems, AF960) and rabbit anti-Vimentin (Abcam, AB92547). Secondary antibodies (1:1000): donkey anti-goat 488 (Molecular Probes, A-11055) and donkey anti-rabbit 568 (Molecular Probes, A10042). Immuno-stained tails were imaged on a Prairie two-photon system, using an Olympus 20 × 1.0 NA W objective, with the excitation laser tuned to 960 nm, and GaAsP photodetectors. Z stacks of 1024 × 1024 images were acquired every 1 μm, with either 1x or 1.5x zoom. Laser intensity and photomultiplier levels were maintained across replicates and controls.
8
Immunofluorescence Staining of Eye Tissues
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Immunofluorescence staining was performed on the eye sections of the animals to examine the location and expression of different types of VEGFs, and the eyes were collected following euthanasia and cryostat-sectioned at 5 μm thickness. Each section was mounted on a slide and prepared for immunohistochemical staining. Anti-VEGF and anti-PlGF were used as the primary antibodies, and Alexa 488 (A11055, Molecular Probes, Life Technologies, Grand Island, NY, USA) and Alexa 555 (A31572, Molecular Probes) were used as the secondary antibodies. Isolectin IB4 conjugated Alexa 594 (121413, Molecular Probes, Life Technologies, Grand Island, NY, USA) was used to stain the endothelial cells. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, sc-3598, Santa Cruz Biotechnology). An upright fluorescence microscope (DM2500, Leica) with a cooled Charge-coupled Device (CCD) camera system (CoolSNAP EZ, Roper Scientific, Martinsried, Germany) was used to observe epifluorescence expression of the retinal sections.
9
Immunostaining of Rat Spinal Cord Sections
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To anesthetize rats, pentobarbital (100 mg/kg, i.p.) was administered. Fully anesthetized rats were perfused transcardially with PBS, followed by ice-cold 4% paraformaldehyde/PBS. The L4 segments of the spinal cord were removed, postfixed in the same fixative for 3 h at 4 °C, placed in 30% sucrose solution for 24–48 h at 4 °C, embedded by Tissue-Tek O.C.T. Compound (Sakura Finetek Japan, Tokyo, Japan) and stored at −80 °C before use. The L4 spinal cord transverse sections (30 μm) were made and were immunostained by the free-floating method as described previously [9 (link),32 (link)]. The sections were incubated in blocking solution for 2 h and followed by the primary antibodies: polyclonal goat anti-paired box 2 (PAX2; 1:500; AF3364, R&D Systems, Minneapolis, MN, USA) and isolectin B4 (IB4) biotin-conjugate (1:1000; I21414, Thermo Fisher Scientific, Waltham, MA, USA) for 48 h at 4 °C. The sections were washed and incubated with secondary antibodies conjugated with streptavidin-conjugated Alexa Fluor 405 and Alexa Fluor 488 (1:1000; S32351 and A11055, Molecular Probes, Eugene, OR, USA) for 3 h. We then analyzed the sections using a confocal microscope (LSM700, Zeiss, Oberkochen, Germany).
10
Quantifying Oxidative Nucleic Acid Damage
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Oxidative nucleic acid damage was assessed by 8-OHdG immunofluorescence in the aorta sections. After deparaffinization and hydration, sections were heat treated (1 mmol/l EDTA, pH 8.0) for 15 min in a microwave. Primary 8-hydroxyguanosine antibody incubation (8-OHG, 1:200, Ab10802; Abcam, Cambridge, UK) was performed overnight at 4°C, followed by rinsing in TBS+ 0.05% Tween20, and incubation with secondary antibody (Alexa-fluor-488-conjugated, 1:300, A-11055; Molecular Probes, Eugene, Oregon, USA). Lipofuscin-mediated autofluorescence was removed by using 0.1% Sudan Black B (Sigma, St Louis, Missouri, USA). Slides were mounted using ProLong Gold Anti-fade mounting media containing DAPI (Molecular Probes) for nuclei staining. All sections were analysed in Live Cell Microscope (Zeiss, Cambridge, UK), using fluorescent filters (Alexa-fluor-488 and DAPI), and obtained images for each fluorescent filter. 8-OHG analysis was performed by measuring the intensity of immunofluorescence in Alexa-fluor-488 [CTTF = Integrated Density – (Area of selected tissue X Mean fluorescence of background readings].
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