For immunostaining, human iPSCs and EBs were fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature, washed with PBS, and permeabilized in 0.3% Triton X-100 in PBS for 10 min at room temperature. Primary antibodies used were anti-Oct4 (ab18976, Abcam, Cambridge, UK), anti-Nanog (ab62734, Abcam), anti-SOX2 (ab97959, Abcam), anti-TRA-1-60 (ab16288, Abcam), anti-Nestin (ab22035, Abcam), anti-α-SMA (ab5694, Abcam), anti-Brachyury (AF2085, R&D Systems, Minneapolis, MN, USA), and anti-GATA4 (ab134057, Abcam). After primary antibody incubation, samples were washed with PBS and incubated with secondary Alexa Fluor 488-conjugated antibodies (Life Technologies), diluted 1:2,000. Samples were also counterstained with DAPI (200 μg/mL). Slides were observed using an Olympus BX61 research microscope equipped with a cooled CCD camera. Images were captured and analyzed with Applied Imaging software CytoVision.
Af2085
Manufactured by R&D Systems
Sourced in United States
AF2085 is a protein detection reagent used in immunoassay applications. It serves as a secondary antibody that binds to the primary antibody, enabling detection and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Af1924, by R&D Systems (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Sc 5279, by Santa Cruz Biotechnology (3 mentions)
Af1997, by R&D Systems (3 mentions)
Mab5326, by Merck Group (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Axio observer a1, by Zeiss (3 mentions)
Ab22035, by Abcam (2 mentions)
Ab62734, by Abcam (2 mentions)
Ab35842, by Abcam (2 mentions)
Mab4304, by Merck Group (2 mentions)
Ab5603, by Merck Group (2 mentions)
A21447, by Thermo Fisher Scientific (2 mentions)
A21206, by Thermo Fisher Scientific (2 mentions)
Ab23345, by Abcam (2 mentions)
Ab5790, by Abcam (2 mentions)
Af1924, by Abcam (2 mentions)
M0851, by Agilent Technologies (2 mentions)
Triton x 100, by Merck Group (2 mentions)
26 protocols using af2085
1
Immunostaining of Human iPSCs and EBs
2
Immunocytochemistry Antibody Concentrations
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Immunocytochemistry was performed as described previously (Osorno et al., 2012 (link)). Primary antibody concentrations were: anti-Nanog, 2.5 μg/ml (14-5761-80, eBioscience); anti-Oct4, 1 μg/ml (N-19, Santa Cruz); anti-Sox2, 0.5 μg/ml (Y-17, Santa Cruz) or 1:200 (ab92494, Abcam); anti-Foxa2, 2 μg/ml (M-20, Santa Cruz) or 1:200 (ab40874, Abcam); anti-Gsc 2 μg/ml (N-12, Santa Cruz); anti-T (Bra), 1 μg/ml (AF2085, R&D); anti-Cdh1, 0.4 μg/ml (ECCD2, Calbiochem); anti-Cdh2, 5 μg/ml (8C11, BD Biosciences); anti-Nes, 1:20 (Rat-401, DSHB); anti-Nkx2.5, 20 μg/ml (ab35842, Abcam); anti-CD31, 4 μg/ml (MEC 13.3, BD Pharmingen).
3
Pluripotency and Germ Layer Characterization of hc-iPSCs
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The putative hc-iPSCs were fixed with 4% paraformaldehyde (PFA) in DPBS for 20 minutes for immunofluorescent staining, following our routine protocol [22 (link)]. Primary antibodies against OCT4 (1:150, MAB4401, Millipore), SOX2 (1:150, GTX101507, Genetex, Hsinchu, Taiwan), SSEA4 (1:150, MAB4304, Millipore), TRA1-60 (1:200, MAB4360, Millipore) and TRA-1-81 (1:200, MAB4381, Millipore) were used for detecting pluripotency, and those against SOX17, BRACHYURY (1:50. AF1924 and AF2085, R&D Systems Inc., Minneapolis, MN, USA) and β-III-TUBULIN (or TUJ1, 1:200, MAB1637, Millipore) were used for detecting germ layers differentiation. Secondary antibodies include Alexa Fluor goat anti mouse 488 (A11029), goat anti rabbit 488 (A11008), and donkey anti mouse IgM Cy3 (715-165-140, Jackson ImmunoResearch Inc., West Grove, PA, USA). Fixed samples were also subjected to alkaline phosphatase (AP) detection by VECTOR Blue Alkaline Phosphatase Substrate Kit (SK-5300, Vector Laboratories, Burlingame, CA, USA). Teratoma tissues were dissected and immersed in 4% PFA overnight at 4°C and then embedded into wax. Sections were dewaxed, rehydrated and stained with hematoxylin and Eosin (H&E).
4
Differentiation of iPSCs into Lineages
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iPSCs at passages 32 to 38 were differentiated for 7 days on 8-well Ibidi µ-slides using an adapted protocol from13 (link). Cells differentiated for 0, 3, and 7 days were fixed in 4% (v/v) paraformaldehyde for 10 min at RT and blocked for 1 h at RT using blocking solution containing 5% (v/v) goat or donkey serum. Cells were incubated in primary antibody at 4 °C overnight. Following incubation, cells were washed with DPBS and incubated overnight at 4 °C with Hoechst33342, and their corresponding fluorescence-conjugated secondary antibody (1:200-500). Images were acquired using a Leica SP8 confocal microscope. Antibodies and dilutions used were OCT4 (1:300; sc-5279, Santa Cruz), Brachyury (1:100; AF2085, R&D), HOXD11 (1:200; SAB1403944, Sigma), PAX2 (1:200; 71-6000, Invitrogen).
5
Immunofluorescence Analysis of Stem Cell Markers
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Cells were fixed by adding formaldehyde to a final concentration of 3.7% in PBS, then permeabilized and blocked in PBS/0.1% TritonX-100/4% (w/v) BSA. Incubation was performed at 4°C overnight with primary antibodies at the following concentrations: goat anti-brachyury 1 µg ml−1 (AF2085, R&D), rabbit anti-Sox2 5 µg m−1 (ab5603, Millipore), rabbit anti-β-III-tubulin 1 µg ml−1 (T2200, Sigma-Aldrich), mouse anti-HB9 1.75 µg ml−1 (81.5C10, Developmental Studies Hybridoma Bank) and rabbit anti-islet 1 2.5 µg ml−1 (ab20670, Abcam). Fluorochrome-conjugated secondary antibodies used were the following: anti-goat Alexa647-conjugated 4 µg ml−1 (A21447, Invitrogen), anti-rabbit Alexa488-conjugated 4 µg ml−1 (A21206, Molecular Probes) and anti-mouse Alexa594-conjugated 4 µg ml−1 (A11032, Molecular Probes). Observations were carried out with a DeltaVision fluorescence microscope (GE Healthcare) and images were acquired using softWoRx software, except images in Fig. S8, which were captured on a Zeiss LSM 710 confocal microscope.
6
Immunofluorescence Assay for Pluripotency and Lineage Markers
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For the detection of pluripotency and differentiation markers, cells grown in 24-well plates as described above were fixed with 4% paraformaldehyde for 20 min. After that, cells were permeabilized and blocked with 10% donkey serum and 0.1% Triton X-100 in PBS. Subsequently, cells were stained with primary antibodies overnight at 4°C and finally incubated with Fluorochrome-labeled secondary antibodies ALEXA FLUOR (Invitrogen). The primary antibodies used for detecting pluripotency markers were anti-OCT4 (SC-5279, Santa Cruz Biotech), anti-SOX2 (AF2018, R&D), and anti-NANOG (AF1997, R&D). The primary antibodies used for detecting endoderm markers were anti-SOX17 (AF1924, R&D), anti-CXCR4 (MAB173-100, R&D), and anti-GATA4 (SC-25310, Santa Cruz Biotech). The primary antibodies used for detecting mesoderm markers were anti-Brachyury (AF2085, R&D), anti-EOMES (Ab23345, Abcam), and anti-MIXL1 (SC-98664, Santa Cruz Biotech). The primary antibodies used for detecting neuroectoderm markers were anti-NESTIN (AB22035, Abcam), and anti-SOX1 (AF3369, R&D) anti-SOX2 (AF2018, R&D). The secondary antibodies used were donkey anti-goat AF488 (Invitrogen), donkey anti-mouse AF488 (Invitrogen), and donkey anti-rabbit AF488 (Invitrogen). Images were captured and quantified using a Cellomics array scan, and images were processed using Adobe Photoshop CS5.
7
Immunofluorescence Staining of SOX1 and T
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Immunofluorescence staining was performed as previously described (Murakami et al, 2016 ). The following primary antibodies were used: rabbit anti‐SOX1 (1:200, Cell Signaling, 4194) and goat anti‐T (1:200, R&D Systems, AF2085).
8
Pluripotent Stem Cell Characterization
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Human ESCs and iPSCs were cultured on cover glass and fixed by incubating with 4% paraformaldehyde for 20 min at room temperature (RT). Fixed cells were then washed with PBS and permeabilized using a non-ionic detergent (0.1% Triton X-100 and 0.2% Tween-20) in PBS for 40 min at RT. Permeabilized cells were blocked by incubating with 2% goat serum (Invitrogen) for 1 h, washed with PBS containing 0.01% Tween-20 (PBST), and incubated with primary antibody. Primary antibodies used included anti-SOX17 (1:200; R&D Systems; AF1924), anti-PAX6 (1:500; Abcam; Ab5790), anti-NANOG (1:50; Abcam; Ab21624), anti-OCT4 (1:1000; Millipore; MAB4401), and anti-Brachyury (1:50; R&D Systems; AF2085). Cells were then washed with PBST and incubated with the appropriate fluorescein-conjugated secondary antibody. Stained samples were mounted using Vectashield H-1200 mounting media (Vector Laboratories), and images were captured using a fluorescence microscope (Leica). Positive signals in IF images were processed and counted using Image J with a consistent intensity threshold.
9
Immunofluorescence Analysis of Pluripotency and Lineage Markers
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OCT4: Santa Cruz Biotechnology (sc-5279), 1:200
BRA: R&D Systems (MAB20851-100), 1:500
BRA: R&D Systems (AF2085), 1:300
GATA6: R&D Systems (AF1700), 1:200
GATA6: Cell Signaling (D61E4), 1:200
GATA3: Thermo Fisher Scientific (14-9966-82), 1:100
SOX17: R&D Systems (MAB1924), 1:200
HAND1: R&D Systems (AF3168-SP), 1:200
KRT7: Abcam (ab209600), 1:100
ISL1: DSHB (39.4D5), 1:200
MIXL1: Sigma Prestige antibody (HPA005662), 1:200
SUSD2: Miltenyi Biotec (130-106-401), 1:100
SSEA4: Miltenyi Biotec (130-122-958), 1:100
KLF17: Atlas Antibodies (HPA024629), 1:200
SOX17: R&D Systems (AF2864), 1:200
GATA4: Thermo Fisher Scientific (14-9980-82), 1:100
FOXA2: Novus Biologicals (AF2400), 1:200
NR2F2: Abcam [EPR18442] (ab211776), 1:100
CD24: eBioscience (A5-2H10), 1:100
Vimentin: Abcam (ab8978), 1:200
10
Immunostaining of Pluripotent Stem Cells
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Cells for immunostainings were cultured on eight-chamber µ-slides (ibidi) and processed as previously described (Schröter et al., 2015 (link)). Primary antibodies used were anti-NANOG 1:200 (Thermo Fisher Scientific, eBioMLC-51), anti-T/BRA 1:200 (R&D Systems, AF2085), anti-HAND1 1:200 (R&D Systems, AF3168), anti-TBX6 1:200 (R&D Systems, AF4744) and anti-CDX2 1:250 (BioGenex, MU392A-5UC). Secondary antibodies coupled with appropriate AlexaFluor dyes were from Thermo Fisher Scientific and used at 1:500 dilution.
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