Sox1‐GFP+ cells were measured at day 0, 2, 4, and 10 using the Aria II platform and normalized to day 0 (Figure
Ab19857
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab19857 is an antibody product manufactured by Abcam. It is a recombinant monoclonal antibody targeting a specific protein. The core function of this product is to specifically bind and detect the target protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab97959, by Abcam (16 mentions)
Ab80892, by Abcam (11 mentions)
Ab21624, by Abcam (7 mentions)
Ab16285, by Abcam (6 mentions)
Ab92494, by Abcam (5 mentions)
Ab16287, by Abcam (5 mentions)
Ab109250, by Abcam (4 mentions)
Ab18465, by Abcam (4 mentions)
Ab13970, by Abcam (3 mentions)
Af1997, by R&D Systems (3 mentions)
Triton x 100, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Ab22035, by Abcam (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Ab16051, by Abcam (2 mentions)
Ab150075, by Abcam (2 mentions)
M0851, by Agilent Technologies (2 mentions)
Nb600 502, by Novus Biologicals (2 mentions)
A11005, by Thermo Fisher Scientific (2 mentions)
92 protocols using ab19857
1
Quantification of Sox1-GFP+ Cells
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Sox1‐GFP+ cells were measured at day 0, 2, 4, and 10 using the Aria II platform and normalized to day 0 (FigureS1 C). For Figure S1 B, cells were fixed for 10 minutes with ice‐cold paraformaldehyde and blocked for 30 minutes at 24°C with 0.1% Triton X100‐1% BSA in PBS. Anti‐Oct4 (ab19857, Abcam, Cambridge, Cambridgeshire, United Kingdom, ab19857.html">https://www.abcam.com/oct4-antibody-chip-grade-ab19857.html ), anti‐Nanog (ab80892, Abcam, Cambridge, Cambridgeshire, United Kingdom, ab80892.html">https://www.abcam.com/nanog-antibody-ab80892.html ), anti‐Tubb3 (ab78078, Abcam, Cambridge, Cambridgeshire, United Kingdom, ab78078.html">https://www.abcam.com/beta-iii-tubulin-antibody-2g10-neuronal-marker-ab78078.html ), and anti‐GFP (ab13970, Abcam, Cambridge, Cambridgeshire, United Kingdom, ab13970.html">https://www.abcam.com/gfp-antibody-ab13970.html ) (all at 1:1000) were used, with DAPI as nuclear counterstain (Life Technologies, D1306).
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Sox1‐GFP+ cells were measured at day 0, 2, 4, and 10 using the Aria II platform and normalized to day 0 (Figure
2
Immunostaining of Adherent Cells
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Cells cultured on gelatin-coated glass-bottom dishes were fixed with 4% paraformaldehyde in PBS at 4°C overnight, followed by 3× washes in wash buffer (0.1% Triton X-100 in PBS). Cells were incubated in blocking buffer (PBS with 1% BSA and 0.1% Triton X-100), and then incubated with primary antibodies diluted in blocking buffer at 4°C overnight. After washing with wash buffer, cells were incubated with secondary antibodies diluted with blocking buffer at room temperature for 1 h, and mounted with 40% glycerol in PBS containing 2% 1,4-Diazabicyclo [2.2.2] octane (DABCO) (Sigma, D2522). Images were captured using a LEICA AF6500 fluorescence imaging system (Leica). Antibodies used in this research were as follows: primary antibodies—biotinylated CDC324 (E-cadherin: CDH1) rat monoclonal antibody (Thermo Fisher Scientific, 13-3249-82) (1:200) and anti-OCT4 rabbit polyclonal antibody (Abcam, ab19857) (1:400); and secondary antibodies—Alexa Fluor 488 anti-rat donkey IgG (Thermo Fisher Scientific, A21208) (1:500) and Alexa Fluor 555 anti-rabbit donkey IgG (Thermo Fisher Scientific, A31572) (1:500).
3
Immunofluorescence Characterization of ESCs and PGCLCs
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The collected ESCs and PGCLCs formed in different induction systems were fixed with 4% paraformaldehyde for 30 min, washed with PBS three times and treated with 1% Triton X-100 for 15 min, followed by the addition of 10% FBS to block the cells for 2 h. Then, the primary antibodies against SSEA-1 (Abcam, Cambridge, MA, USA, ab16285), OCT4 (Abcam, ab19857) and CVH (Abcam, ab13840) were added and incubated at 37 °C for 2 h and then at 4 °C overnight. After washing three times with PBST, the cells were incubated with the corresponding secondary antibodies at 37 °C for 2 h without light, and then washed three times with PBST, followed by incubation with DAPI for 10 min without light, and then washed again. Fluorescence was observed and photographed under the fluorescence microscope.
4
Protein Expression Analysis of Stem Cells
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The total protein of tissues and cells was extracted. After ice bathing and centrifugation, the sample was added to each lane for electrophoresis separation. The separated protein was transferred onto a nitrocellulose membrane. Then, the nitrocellulose membrane was blocked with 5% skim milk powder at 4°C overnight and incubated overnight with primary rabbit anti‐mouse polyclonal antibodies diluted at the ratio of 1:1000: NEK2 (ab115731), β‐catenin (ab16051), Nanog (ab21624), Oct‐4 (ab19857), CXCL16 (ab101404), EGFR (ab52894), TCF‐4 (ab217668) and Pygo2 (ab109001) and rabbit anti‐mouse monoclonal antibodies diluted at 1:1000: CD24 (ab64064), and CD44 (ab51037), all of which were purchased from Abcam Inc, Cambridge, UK. The membrane was washed 3 times with PBS at room temperature, 5 minutes for each, incubated with oscillation with the addition of the secondary antibody of horseradish peroxidase (HRP)‐labelled goat anti‐rabbit immunoglobulin (IgG) (1:10 000, ab6728, Boster Biological Technology Co. Ltd, Wuhan, China) at 37°C for 1 hour, washed with Tris‐buffered saline‐Tween (TBST) and visualized using HRP electroluminescence (ECL). Grey value analysis of the target bands was performed using ImageJ software, and the experiment was repeated three times independently.
5
Immunofluorescence Staining of Cultured mESCs
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Monolayer cultured mESCs were fixed in situ in 4% w/v PFA in PBS for 30 minutes at room temperature, washed twice with PBS and blocked with 1% goat serum in 0.1% w/v BSA, 0.1% Triton X-100 in PBS for 30 minutes. Cells were incubated in the appropriate primary antibody in goat blocking buffer for two hours at RT. Cells were washed three times in PBS and incubated in the appropriate Alexa fluor-488 or −546 conjugated secondary antibody at RT for 1 hour in the dark. Cells were washed three times with PBS, mounted in 4′6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) and visualized using a Leica DM6000B fluorescence microscope (Leica Microsystems) and processed using Leica LAS X Core software (Leica Microsystems). Primary antibodies: anti-Nanog, 1:200 dilution (ab80892, Abcam Plc, Cambridge, UK); anti-Oct4, 1:200 dilution (ab19857, Abcam Plc, Cambridge, UK). For immunofluorescence microscopy analysis of fluorescent-tagged peptides, cells were incubated in 500 μM peptide in cell culture medium for 15 minutes, washed twice in ice cold PBS and mounted with Vectorshield containing DAPI. Cells were visualized using a Leica DM6000B fluorescence microscope (Leica Microsystems) and processed using Leica LAS X Core software (Leica Microsystems).
6
Chromatin Immunoprecipitation Profiling of OCT4
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ChIP was performed using 10 E9.5 embryos or 1 × 106 ES cells per experiment. After recovery, embryos were treated with collagenase type I (Stemcell Technologies, 07902) at 0.125% for 1 hour at 37°. Then, embryos were desegregated using a pipette and washed with cold phosphate-buffered saline. Samples were fixed, and protein-DNA complexes were cross-linked by treatment with 1% formaldehyde (Pierce, 289069) for 15 min rocking at room temperature. To stop fixation, glycine (Nzytech, MBO1401) was added to a final concentration of 125 mM during 10 min. Next, ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif, 53040), following the manufacturer’s instructions. DNA was sheared into fragments ranging from 200 to 1000 bp using a sonicator (Diagenode Bioruptor Water Bath Sonicator, 30 s on 30 s off for 30 min). Immunoprecipitations were carried out using rabbit polyclonal anti-OCT4 antibody (Abcam, ab19857), and anti-Rabbit immunoglobulin G polyclonal antibody (Abcam, ab171870) was used as negative control. Enrichment was measured by qPCR. A fragment from the Nanog promoter was used as a positive control (27 (link), 29 (link)), and genomic fragments from the loci of Anks1b, Smg6, and Tiam1 were used as negative controls (59 (link)) after checking they did not contain OCT4 bound peaks (27 (link)). qPCR primers used are listed in data file S4.
7
Antibody Characterization for Stem Cell Analysis
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Primary antibodies used were as follows: mouse anti-SSEA-4 (1:100, MAB4304, Millipore), goat anti-Nanog (1:20, AF1997, R&D Systems), rabbit anti-Oct4 (1:200, ab19857, Abcam), mouse anti-α-SMA (1:200, M0851, Dako), goat anti-Sox17 (1:200, AF1924, R&D Systems), rabbit anti-Tuj1 (1:2000, MRB-435P, Covance), mouse anti-Flag M2 (ICC, 1:500; WB, 1:100, F1804, Sigma), mouse anti-HA Tag (6E2) (ICC, 1:100; WB, 1:1000, #2367, Cell Signaling), rabbit anti-Tom20 (1:100, sc-11415, Santa Cruz), and mouse anti-GAPDH (1:1000, NB600-502, Novusbio). Alexa Fluor 488 (A11055 or A11034, Molecular Probes), Alexa Fluor 594 (A11005, Molecular Probes) and Alexa Fluor 647 (ab150075, Abcam)-conjugated secondary antibodies were used for immunofluorescent study.
8
Immunofluorescence Staining of Cells and Tissues
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Cells or tissues were stained per the manufacturer’s protocol using rabbit polyclonal (ab19857, Abcam, Cambridge, MA; 1:200 dilution) or murine anti-OCT-4 (ab91194, Abcam, Cambridge, MA; 1:200 dilution), and rat anti-Vimentin (ab115189, Abcam, Cambridge, MA; 1:200 dilution). Secondary antibodies were Allophycocyanin anti-mouse IgG (A-865, Life Technologies, Grand Island, NY; 1:1000), AlexaFluor 555 anti-rabbit IgG, and anti-Rat IgG AlexaFluor 647 (Abcam, Cambridge, MA; 1:1000 dilution). Cells were visualized with an Olympus FV1000 Confocal Microscope using 488 nm, 543 nm, or 633 nm for excitation. Tissue was visualized with an Olympus BX51 upright epifluorescence microscope (Olympus America, Center Valley, PA) using excitation/emission filters of 480 nm/535 nm, and 620 nm/700 nm.
9
Analysis of SORE6-driven GFP Expression
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The level of SORE6-driven GFP fluorescence in untreated cultures or after different drug treatments were analyzed and/or SORE6+ and SORE6- subpopulations were sorted by flow cytometry using a BD FACS Aria II Cell Sorter (BD Bioscience, Erembodegem, Belgium). Cells transduced with the minCMVp-GFP lentivirus were used as matched SORE6 negative control for gating purposes. In these analyses, dead cells were excluded by propidium iodide (0.5 μg/mL) staining (Figure S8 ). To analyze the induction of apoptosis in SORE6+ and SORE6- subpopulations, unfixed cells were assayed for active caspase-3 immediately after treatment using the PE Active Caspase-3 Apoptosis Kit (BD Bioscience) according to the manufacturer’s instructions and the level of GFP (SORE6) and PE (Caspase 3) fluorescence was simultaneously detected by flow cytometry. SOX2 and OCT4 expression was detected by flow cytometry in 70% ethanol-fixed cells using an anti-SOX2 antibody from Thermo Fisher (Waltham, MA) (PA1-094); 1: 1000 dilution) or anti-OCT4 antibody from AbCam (Cambridge, UK) ((ab19857); 1: 1000 dilution).
10
Adipose-derived Stem Cells Differentiation into Neural Stem Cells
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ADSCs differentiate into NSCs by neurosphere technique. In the first stage, ADSCs of the fourth passage were removed using trypsin and EDTA and plated with serum‐free DMEM/F12 containing 2% B27, 20 ng/mL of the epidermal growth factor (EGF), and 20 ng/mL of the basic fibroblast growth factor (bFGF) (Invitrogen, Paisley, Scotland). After 7 days, the cell aggregates (neurosphere) were harvested into single cells and the dissociated cells were then cultured in a T25 flask (2 × 106 density) in a neurosphere medium with 10% FBS (NSC culture medium). NSCs were cultured on a six‐well plate and immunolabeled with primary antibodies against nestin (Abcam, ab11306), NF 68 (Abcam, ab223343), Neurod (Abcam, ab239955), Sox2 (Abcam, ab92494), Oct4 (Abcam, ab19857), and Neun (Abcam, ab177487) (Darvishi et al., 2020 (link); Moayeri et al., 2020 (link)).
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