Cells were washed twice in PBS, then fixed in freshly prepared 3.7% paraformaldehyde in PBS (pH 7.4) for 30 min at 4 °C, washed in PBS for one time and permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum plus 0.1% BSA in PBS) for 30 min at room temperature, then washed in PBS for one time, and left in blocking solution for 2 h. Cells were incubated overnight at 4 °C with primary antibodies against Oct4 (sc5279; Santa Cruz), Nanog (ab80892; Abcam), SSEA-1 (MAB4301; Millipore), βIII-tubulin (CBL412; Chemicon), alpha 1-fetoprotein (AFP; DAK-N1501; Dako), alpha smooth muscle actin (α-SMA; ab5694-100; Abcam), γH2AX (05-636; Millipore), TRF1 (TRF12-S; Alpha Diagnostic) and Zscan4 (AB4340; Millipore). Then cells were washed three times (each for 15 min) with blocking solution, and incubated for 2 h with secondary antibodies at room temperature. Goat Anti-Mouse IgG (H + L) FITC (115-095-003; Jackson) and Goat Anti-Rabbit IgG (H + L) Alexa Fluor® 594 (111-585-003; Jackson), diluted 1:200 with blocking solution, were used. Samples were washed, and counterstained with 0.5 μg/ml Hoechst 33342 (H1398; MP) in Vectashield mounting medium. Fluorescence was detected and imaged using a Zeiss Axio-Imager Z1 fluorescence microscope.
Ab4340
Manufactured by Merck Group
Sourced in United States
The AB4340 is a laboratory equipment product manufactured by Merck Group. It is a device used for performing various scientific experiments and analyses. The core function of the AB4340 is to provide a controlled environment for conducting experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab8898, by Abcam (3 mentions)
Ab1791, by Abcam (3 mentions)
Ab13604, by Abcam (2 mentions)
Ab13888, by Abcam (2 mentions)
P30002, by Abmart (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Ab2893, by Abcam (2 mentions)
Ha sc 7392, by Santa Cruz Biotechnology (2 mentions)
Sc 5279, by Santa Cruz Biotechnology (2 mentions)
γh2ax, by Merck Group (2 mentions)
Mab4301, by Merck Group (1 mentions)
Cbl412, by Merck Group (1 mentions)
Dak n1501, by Agilent Technologies (1 mentions)
Ab5694 100, by Abcam (1 mentions)
Goat anti mouse igg h l fitc, by Jackson ImmunoResearch (1 mentions)
Goat anti rabbit igg h l alexa fluor 594, by Jackson ImmunoResearch (1 mentions)
Axio imager z1 fluorescence microscope, by Zeiss (1 mentions)
Ab80892, by Abcam (1 mentions)
Wbkls0500, by Merck Group (1 mentions)
Ab22551, by Abcam (1 mentions)
14 protocols using ab4340
1
Immunofluorescence Analysis of Stem Cell Markers
2
Western Blot Analysis of Epigenetic Markers
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Cells were washed twice in PBS, collected, and lysed in cell lysis buffer on ice for 30 min and then sonicated for 1 min at 60 of amplitude with 2 s intervals. After centrifugation at 10,000g, 4 °C for 10 min, supernatant was transferred into new tubes. The concentration of the protein sample was measured by bicinchoninic acid, and then protein samples were boiled in SDS Sample Buffer at 99 °C for 10 min. 20 μg or 40 μg (for Tcstv1/3 and Zscan4) total proteins of each cell extracts were resolved by 10% or 12% (for Tcstv1/3) Bis-Tris SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF; Millipore). Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 2 h. Blots were then probed with primary antibodies, Tcstv1/3 (custom-made), Zscan4 (AB4340; Millipore), H3K9me3(ab8898; Abcam), H3K9Ac (04-1003; Millipore), H3Ac (06-599; Millipore), H3 (ab1791; Abcam), Dnmt3a (ab13888; Abcam), Dnmt3b (ab13604; Abcam) and β-actin (P30002; Abmart) by overnight incubation at 4 °C in 5% skim milk in TBST. Immunoreactive bands were then probed for 2 h at room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, anti-Rabbit IgG-HRP (GE Healthcare, NA934V), or goat anti-Mouse IgG (H + L)/HRP (ZSGB-BIO, ZB-2305). Protein bands were detected by Chemiluminescent HRP substrate (Millipore, WBKLS0500).
3
Zscan4 and γH2A.X Immunostaining of 2C Embryos
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For Zscan4 and γH2A.X immunostaining, fixed 2C embryos were incubated with anti-Zscan4 (1:200; AB4340, Millipore) and anti-γH2A.X (1:250; ab22551, Abcam) for 3 to 4 hours at RT. Followed by several washes in blocking solution, embryos were incubated at RT with anti-mouse and anti-rabbit secondary antibodies for 1.5 hours coupled with Alexa Fluor 488, 594, or 568 (1:500; Invitrogen), respectively. EU staining was performed using the Click-It RNA Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific). Embryos were washed and mounted on slides with a small drop of VECTASHIELD (VectorLab) mounting medium. Mounted embryos were analyzed on a Zeiss LSM510 Meta inverted laser scanning confocal microscope and computations of z-stack images were processed as described previously (40 (link)). ImageJ software was used to quantify Zscan4 antibody signals and γH2A.X foci of z-stack computed (~16 inner stacks with 0.3 μm per sample) Immunofluorescence images as previously described (39 (link)).
4
Western Blot Analysis of Cellular Proteins
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For western with IP samples (see previous session), we included the input control which consists of 1% cell lysate. For regular western, 30 µg protein lysate from each sample was used.
Samples were run in electrophoresis using 10% acrylamide gels and then transfer to 0.22 µm PVDF membrane (GE10600021, Millipore). 5% skim milk in TBST (0.1% Tween 20 in TBS) was used to block the membrane for 30 min at room temperature. The membrane was immunoblotted with the primary antibody overnight at 4 ℃ then incubated with HRP-conjugated secondary anti-mouse antibody (31430, Thermo) or HRP-conjugated secondary anti-rabbit antibody (31460, Thermo) for 2 h at room temperature. The signal was detected by T-Pro LumiFast Plus Chemiluminescent Substrate Kit (JT96-K002, T-pro, New Taipei, Taiwan) and captured by GeneGnome XRQ Chemiluminescence with CCD (SynGene, Cambridge, UK). The primary antibodies included FLAG antibody (F7425, MilliporeSigma), HA (sc-7392, Santa Cruz), γH2AX (ab2893, Abcam), ZSCAN4 (ab4340, Millipore), and PARP1 (9542, Cell Signaling, Danvers, MA, USA).
Samples were run in electrophoresis using 10% acrylamide gels and then transfer to 0.22 µm PVDF membrane (GE10600021, Millipore). 5% skim milk in TBST (0.1% Tween 20 in TBS) was used to block the membrane for 30 min at room temperature. The membrane was immunoblotted with the primary antibody overnight at 4 ℃ then incubated with HRP-conjugated secondary anti-mouse antibody (31430, Thermo) or HRP-conjugated secondary anti-rabbit antibody (31460, Thermo) for 2 h at room temperature. The signal was detected by T-Pro LumiFast Plus Chemiluminescent Substrate Kit (JT96-K002, T-pro, New Taipei, Taiwan) and captured by GeneGnome XRQ Chemiluminescence with CCD (SynGene, Cambridge, UK). The primary antibodies included FLAG antibody (F7425, MilliporeSigma), HA (sc-7392, Santa Cruz), γH2AX (ab2893, Abcam), ZSCAN4 (ab4340, Millipore), and PARP1 (9542, Cell Signaling, Danvers, MA, USA).
5
Protein Expression Analysis of Stem Cell Markers
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Cells were washed twice in PBS, collected, lysed, and boiled in SDS sample buffer at 99 °C for 5 min; Equal amounts of total proteins of each cell extracts were resolved by 10–12% Bis-Tris SDS-PAGE and transferred to polyvinylidinedifluoride membranes (PVDF, Millipore, Burlington, MA, USA). Nonspecific binding was blocked by incubation in 5% skim milk in TBST at room temperature for 1–2 h. Blots were then probed with various primary antibodies, anti-Zscan4 (AB4340, Millipore, 1:1000), Dnmt1 (sc10221, Santa Cruz, 1:500), Dnmt3a (ab13888, Abcam, 1:1000), Dnmt3b (ab13604, Abcam, 1:1000), Tet2 (Kind gift from Dr. Jinsong Li from SIBS, 1:1000), H3K9me3 (ab8898, Abcam, 1:2000), H3 (ab1791, Abcam, 1:2000), and β-actin ((P30002, Abmart, 1:5000) by overnight incubation in 5% skim milk in TBST at 4 °C. Immunoreactive bands were then probed for 1–2 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary anti-Rabbit IgG-HRP (GE Healthcare, NA934V, 1:5000), or anti-mouse IgG-HRP (Santa Cruz, sc-2031, 1:5000), or anti-goat IgG-HRP (Santa Cruz, sc-2020, 1:5000). The protein bands were detected by Enhanced ECL AmershamTM prime Western blotting detection reagent (GE Healthcare, RPN2232).
6
Whole-cell Protein Extraction and Immunoblotting
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Whole-cell extracts were prepared by lysing cells and extracting proteins for 30 min at 4°C in a buffer containing 300 mM NaCl, 100 mM tris (pH 8), 0.2 mM EDTA, 0.1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease inhibitor cocktail (Roche). Supernatant was collected after centrifugation and protein concentrations were determined using Bradford reagent (Bio-Rad). Proteins were separated on an SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes followed by immunoblotting with the following antibodies as indicated: Zscan4 (AB4340, Millipore) and GFP (ab290, Abcam).
7
Zscan4 Expression Analysis in ESCs
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ESCs were collected and washed in cold PBS, then fixed in cold 70% ethanol. Cells were permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum in PBS) for 30 min, then washed and left in blocking solution for 1 h. ESCs were incubated with primary antibodies against Zscan4 (AB4340; Millipore), washed three times, and incubated for 1 h with secondary antibodies, 488 goat anti-rabbit IgG (A11008; Invitrogen). Samples were washed three times with PBS and fluorescence activated cell sorting (FACS) analysis was performed using a Flow Cytometer (BD FACS Calibur).
8
Immunofluorescence Staining Protocol
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Cells on cover slides were fixed with 10% formaldehyde (MA-H121-08, Crespellano, Italy). 2% bovine serum albumin (BSA, A9647, MilliporeSigma) and 0.25% Triton-X-100 (X100, MilliporeSigma) in phosphate-buffered saline (PBS, IB3012, Omics Bio, Taipei, Taiwan) was used for permeabilizing cells before they were incubated with the primary antibodies overnight at 4 ℃ followed by secondary antibodies and DAPI for 2 h at room temperature. The antibodies used were ZSCAN4 (ab4340, Millipore), FLAG (F7425, MilliporeSigma), FLAG (66008-4-Ig, Proteintech, Rosemont, IL, USA), γH2AX (ab2893, Abcam, Cambridge, UK), HA (sc-7392, Santa Cruz, Dalla, TX, USA), Alexa anti-mouse 488 (A11001, Thermo), Alexa anti-rabbit 488 (A11034, Thermo), Alexa anti-mouse 594 (A11032, Thermo), and Alexa anti-rabbit 647 (A27040, Thermo). The images were captured by the laser-scanning confocal microscope (TCS SP5 II confocal microscope, Leica, Wetzlar, Germany).
9
Immunofluorescence Analysis of Zscan4 and H3K9me3
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Cells were washed twice in PBS, then fixed in freshly prepared 3.7% paraformaldehyde (PFA) in PBS (pH 7.4) for 15 min on ice cube, permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum plus 0.5% BSA in PBS) for 30 min at room temperature, washed three times (each for 15 min) and left in the blocking solution for 1 h. Cells were then incubated overnight at 4 °C with primary antibodies against Zscan4 (AB4340, Millipore, 1:500), H3K9me3 (ab8898, Abcam, 1:1000), washed three times (each for 15 min), and incubated for 1 h with the secondary antibody Fluor 568 goat anti-rabbit IgG (A11036, MP), diluted 1:200 with blocking solution. Samples were washed, and counterstained with 0.5 µg/mL Hoechst33342 (H1398, MP) in Vectashield mounting medium. Fluorescence was detected and imaged using a Zeiss inverted fluorescence microscope (Axio Imager Z1).
10
Immunocytochemistry of 2C::turboGFP cells
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The 2C::turboGFP cell line was cultured on gelatin-coated coverslips. At 48 h after RA treatment, cells were washed with PBS, fixed with 4% PFA for 10 min at room temperature and, after four washes with PBS, permeabilized with 0.3% Triton X-100 for 10 min at room temperature. After washing with PBS, primary antibodies were incubated overnight at 4 °C, followed by another three washes in PBS. The antibodies used were mouse turboGFP (TA140041, Origene) and rabbit Zscan4 (AB4340, EMD Millipore). Secondary antibodies were incubated for 1 h at room temperature. Mounting was done in Vectashield mounting medium (Vector Labs). Images were acquired using a Leica SP8 confocal microscope.
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