Cells cultured in 2D were fixed with 4% paraformaldehyde (Santa Cruz, SC 281692, Dallas, TX, USA) blocked with a blocking solution comprised of 10% donkey serum and 0.1% Triton X-100 in PBS −/−. The cells were incubated with primary antibodies followed by secondary antibody incubation and 4′,6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence was observed using an Olympus IX73 microscope. The following primary antibodies were used to detect hPSC-associated markers: OCT4/POU5F1 (Abcam, ab19857, Cambridge, UK), NANOG (R&D systems, AF1997, Minneapolis, MN, USA), TRA-1-81 (ReproCELL USA, Inc., 09-001, Beltsville, MD, USA), TRA-1-60 (Millipore, MAB4360, Burlington, MA, USA) and SSEA-4 (Millipore, MAB4304, Burlington, MA, USA). The following primary antibodies were used to detect expression of germ-layer specific markers: SOX17 (R&D systems, AF1924, Minneapolis, MN, USA), FOXA2 (Abcam, Ab108422, Cambridge, UK), NESTIN (R&D systems, MAB1259, Minneapolis, MN, USA), PAX6 (Biolegend, #901301), α-actinin (Sigma, A7811, St. Louis, MO, USA) and SMA (Millipore, CBL171, Burlington, MA, USA).
Mab4304
Manufactured by Merck Group
Sourced in United States
MAB4304 is a monoclonal antibody used as a laboratory research tool. It recognizes and binds to a specific protein target. The core function of MAB4304 is to facilitate the detection and analysis of the target protein in biological samples through various laboratory techniques.
Automatically generated - may contain errors
Lab products found in correlation
Mab4381, by Merck Group (10 mentions)
Mab4360, by Merck Group (10 mentions)
Af1924, by R&D Systems (5 mentions)
Sc 9081, by Santa Cruz Biotechnology (4 mentions)
Mab4303, by Merck Group (3 mentions)
Af1997, by R&D Systems (3 mentions)
Gtx101507, by GeneTex (3 mentions)
Mab4401, by Merck Group (3 mentions)
Sc 5279, by Santa Cruz Biotechnology (3 mentions)
A11034, by Thermo Fisher Scientific (3 mentions)
Mab1637, by Merck Group (3 mentions)
Triton x 100, by Merck Group (3 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)
Ab19857, by Abcam (2 mentions)
Sc 11415, by Santa Cruz Biotechnology (2 mentions)
Af2085, by R&D Systems (2 mentions)
Ab21624, by Abcam (2 mentions)
25 protocols using mab4304
1
Characterization of hPSC Differentiation
2
Pluripotency Marker Characterization
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ALP staining was performed with the ALP substrate (Sigma) after fixation with 4% paraformaldehyde. Immunofluorescence staining was performed using the following primary antibodies: anti-OCT 3/4 (sc-5279, Santa cruz), anti-SSEA3 (MAB4303, Millipore), anti-SSEA4 (MAB4304, Millipore), anti-TRA-1-60 (MAB4360, Millipore), and anti-TRA-1-81 (MAB4381, Millipore). The fluorescence signals were detected using a conventional fluorescence laser microscope equipped with a color charge-coupled device (CCD) camera. The secondary antibodies used were anti-mouse IgG and IgM or anti-rat IgM conjugated with Alexa Fluor 488 (Jackson Immunoresearch).
3
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.
4
Immunocytochemical Characterization of hESCs
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Undifferentiated hESCs were stained using Alkaline Phosphatase (ALP) substrate kit III (Vector Laboratories, Burlingame, CA, USA) following the manufacturer’s instructions as previously described [4 (link)]. Immunocytochemical staining was carried out following the protocol described previously [12 (link)]. Briefly, harvest the attached cells at indicated stages, fix the cells with 4% paraformaldehyde (PFA), permeabilize the cells in 0.4% Triton X-100 (Sigma-Aldrich, Carlsbad, USA) for 20 min at room temperature to present the intracellular antigens (for membrane antigens, this step can be skipped), block the cells with 10% normal goat serum (Vector Laboratories, Burlingame, CA, USA), and then stain cells with antibodies against SSEA4 (stage-specific embryonic antigen 4) (cat no. MAB4304, Millipore, CA, USA, 1:200) and OCT4 (octamer-binding protein 3/4) (cat no. ab19857, Abcam, 1:200). The antibody labeling was visualized using DyLight 488/549-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA, 1:1000). Nuclei were stained with DAPI (2-(4-amidinophenyl)- 6-indolecarbamidine dihydrochloride) (Invitrogen, Carlsbad, CA, USA, 1 μg/ml). Slide observation and image capture were performed with a Zeiss Observer microscope.
5
Pluripotency Marker Expression in iPSCs
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Human iPSC or ESCs were cultured on 6-well plates with glass coverslips. On day 5, media was removed and cells were rinsed with PBS then fixed with 4% paraformaldehyde for 30 min at room temperature (RT). Cells were then washed twice with 0.1% Tween 20 in PBS (PBT) at RT. For OCT-4 primary antibody, cells were permealized with 1% Triton-X 100/PBS for 30 minutes at RT. After blocking with 4% Goat Serum in PBS for 30 minutes at RT, cells were incubated with primary antibodies, rabbit Oct-3/4 antibodies (sc-9081, Santa Cruz) and mouse SSEA-4 (MAB4304, Millipore) at 1∶200 dilution in PBS, in a humidity chamber overnight at 4°C, followed by washing with PBT 3 times at RT, 5 minutes each, and subsequent incubation with conjugated 2° antibodies, Alexa Fluor-488 nm affinity pure donkey anti-rabbit and Alexa Fluor-594 nm affinity pure Donkey anti-mouse (Jackson Immunoresearch), at 1∶300 dilution in PBS for 1 hour at RT. Cells were then washed with PBT 3 times at RT, 5 minutes each and counterstained with 1 µg/mL DAPI in PBS for 5 minutes.
6
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).
7
Pluripotency Marker Expression in Stem Cells
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Cells were fixed in 4% paraformaldehyde and permeabilized with PBS 1X and 0.1% Triton X-100. Primary antibodies to Oct4, Tra1-81, Tra1-61, and SSEA4 were obtained from Millipore (MAB4305, MAB4381, MAB4303, and MAB4304) and were all used at 1:50 dilution and incubated at 4°C overnight. Fluorescent secondary antibodies were obtained from Santa Cruz Biotechnology (anti-mouse Texas Red conjugate, anti-mouse FITC conjugate, anti-rat 8 Texas Red conjugate, and anti-rat FITC conjugate). All secondary antibodies were used at 1:100 dilutions and incubated at room temperature for 1 h, followed by nuclear staining with DAPI.
8
Pluripotency Markers and Chromosome Preparation
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Alkaline phosphatase activity and immunostaining were assayed as described previously [33 (link)]. The antibodies used were directed against: OCT4 (sx-5279; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NANOG (AF1997; R&D Systems, Minneapolis, MN, USA), SOX2 (AB5603; Millipore, Billerica, MA, USA), SSEA-1 (MAB4301; Millipore), and SSEA-4 (MAB4304; Millipore). The fluorescence-labeled secondary antibodies A11034 and A11029 were from Invitrogen (Carlsbad, CA, USA). Nuclei were detected with 0.5 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI, D3571; Invitrogen) for 1 h. Metaphase mitotic chromosomes were prepared using a conventional air-drying technique. GTG staining (G-banding) was performed as described elsewhere [38 (link)].
9
Maintenance of hPSC Undifferentiation in Suspension Culture
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Example 6
hPSCs sphere cells subjected to suspension static culture in mTeSR medium containing deacylated gellan gum at 0.015% or 0.020% (w/v), the maintenance of the undifferentiation property thereof was examined by flow cytometry analysis. In a polystyrene tube in a sphere state, human ES cells (KhES-1) were passaged 3 times, human iPS cells (253G1) were passaged 4 times. The cells were recovered, stained with SSEA4 antibody (# MAB4304, manufactured by Millipore) and TRA-1-60 (# MAB4360, manufactured by Millipore) antibody, which are surface markers showing undifferentiation property of hPSCs, and the positive rate of cell staining with the antibody was evaluated using FACSCantoII (manufactured by Becton, Dickinson and Company). The results are shown in
10
Pluripotent Stem Cell Immunofluorescence
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Cells were fixed with 4% paraformaldehyde (PFA) in DPBS for 20 min. Fixed cells were incubated with the primary antibody in PBS with 2.5% bovine serum albumin and 0.25% Triton-X100 for one hours at room temperature and incubate with primary antibody at 4 °C for overnight. After washing with PBS, samples were incubated with secondary antibody, which is diluted in the same solution as used in primary antibody dilution. One hours after the secondary antibody reaction, samples were washed and mounted with ProLong Antifade Mountanting medium (P36984). Primary antibodies used in this study include: NANOG (1:500, ab21624, Abcam, Boston, MA, USA), OCT4 (1:150, MAB4401, Millipore), SOX2 (1:150, GTX101507, Genetex, Alton Pkwy Irvine, CA, USA), SSEA4 (1:150, MAB4304, Millipore), TRA1-60 (1:200, MAB4360, Millipore), TRA-1-81 (1:200, MAB4381, Millipore). Secondary antibodies (4 μg/mL) were listed as bellow: Alexa Fluor goat anti-mouse 488 and 546 (A11029 and A10036), goat anti-mouse IgM 594 (A21044), goat anti-rabbit 488 (A32731). Images were acquired with confocal microscope (TCS SP5 II, Leica, Wetzlar, Germany).
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