The cells were grown in 48-well plates, washed with PBS, and fixed in 4% paraformaldehyde in PBS for 10 min [33 (link),40 (link)]. The fixed cells were washed with PBS, permeabilized by 0,1% Triton X-100 solution in PBS for 15 min, and incubated with 3% bovine serum albumin (BSA) in PBS for 1 h at RT. The cells were incubated with mouse anti-Oct4 (1:500) (Santa Cruz Biotechnology, Dallas, TX, USA) and rabbit anti-Nanog (1:1000) (Bethyl Laboratories, Montgomery, TX, USA). Antibodies were diluted in PBS solution with 0.1% Tween 20, 3% BSA overnight at 4 °C. After washing the cells with 0.1% Tween 20 in PBS, they were incubated with secondary antibodies in dilution of 1:1000: anti-mouse-Alexa647 and anti-rabbit-Cy3 conjugated (Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at RT. The cells were washed and stained with DAPI in PBS (1:5000), then covered by PBS/sodium azide, and proceeded to an analysis by the EVOS Cell Imaging Systems (Thermo Fisher Scientific, Waltham, MA, USA).
Mouse anti oct4
Manufactured by Santa Cruz Biotechnology
Sourced in United States, China
Mouse anti-Oct4 is a primary antibody that recognizes the Oct4 protein, a transcription factor crucial for the maintenance of pluripotency in embryonic stem cells. This antibody can be used to detect and study the expression of Oct4 in various cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti sox17, by R&D Systems (7 mentions)
Goat anti gata6, by R&D Systems (5 mentions)
Mouse anti cdx2, by BioGenex (5 mentions)
Triton x 100, by Merck Group (4 mentions)
Goat anti gata4, by Santa Cruz Biotechnology (4 mentions)
Axio observer z1, by Zeiss (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Rabbit anti nanog, by Fortis Life Sciences (2 mentions)
Mouse anti neun, by Merck Group (2 mentions)
Mouse anti nanog, by BD (2 mentions)
Donkey anti mouse alexa 555, by Thermo Fisher Scientific (2 mentions)
Anti mouse alexa 488, by Thermo Fisher Scientific (2 mentions)
Goat anti nanog, by R&D Systems (2 mentions)
Mouse anti ssea4, by Abcam (2 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 555 donkey anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Rabbit anti nanog, by GeneTex (2 mentions)
Rabbit anti nanog, by Cosmo Bio (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Goat anti sox2, by R&D Systems (2 mentions)
26 protocols using mouse anti oct4
1
Immunofluorescent Staining of Pluripotency Markers
2
Immunofluorescence Analysis of Cell Markers
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Tissues were fixed by 4% PFA at 4 °C overnight, embedded in OCT compound, frozen in liquid nitrogen, sliced into 10-μm sections and placed onto glass slides that were treated with Blocking One for 30 min at room temperature. Primary antibodies and dilutions used were rabbit anti-GFP AlexaFluor 488 conjugate (1:400, Life Technologies A21311), mouse anti-Oct4 (1:400, Santa Cruz sc-5279), mouse anti-NeuN (1:500, Millipore MAB377), rabbit anti-GFAP (1:500, Biomedical Technologies Inc. BT-575), rabbit anti-CD45 (1:100, Dako M0701), rabbit anti-DDX4 (VASA) (1:400, Abcam ab13840), which were then detected with appropriate secondary AlexaFluor 488, 568 or 647 antibodies. Cells were counterstained with Hoechst 33342 and observed using a Leica TCS SP8 confocal microscope.
3
Immunofluorescence Assay for Pluripotency Markers
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Plates were fixed in 4% PFA for 10 minutes, washed 3 times with PBS, blocked and permeabilized in PBS supplemented with 5% CCS and 0.1% Triton-X 100 (Sigma) (blocking solution) for 10 minutes. 96 well plates were then incubated with mouse anti-Nanog (1:500, BD) or mouse anti-Oct4 (1:200, Santa Cruz Biotechnology) in blocking solution for 30 minutes, washed 3 times with PBS, incubated with donkey anti-mouse Alexa-555 (1:1000, Invitrogen) or anti-mouse Alexa-488 (1:1000, Invitrogen) in blocking solution for 30 minutes, washed three times with PBS and stained with DAPI for three minutes. Cells were then washed with PBS and visualized.
4
Immunofluorescence Staining of iPSCs
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iPSCs were fixed with 4% paraformaldehyde in PBS for 15 min and washed with PBS for 3 times, 5 min each. Cells were then permeabilized and blocked with 0.1% triton X and 2.5% bovine serum albumin in PBS for 1 hour and incubated in primary antibodies overnight at 4 °C (mouse anti-OCT4 from Santa Cruz (cat#SC-5279), 1:250; rabbit anti-LIN28 from Abcam (cat#AB46020), 1:500; mouse anti-SSEA4 from Abcam (cat#MC813), 1:200; rabbit anti-NANOG from GeneTex (cat#GTX100863), 1:300; goat anti-NANOG from R&D (cat#AF1997), 1:250; rabbit anti-H3K27me3 from Millipore (cat#07-449), 1:500); mouse anti-Tra-1-60 from Abcam (cat#AB16288), 1:500; and anti-Tra-1-81 Alexa647 from BD Biosciences (cat#BDB560124), 1:10). After washed with PBS for 3 times, 5 min each, cells were incubated in secondary antibodies if necessary (Alexa Fluor 488 donkey anti-mouse IgG (cat#A21202), 1:500; Alexa Fluor 555 donkey anti-rabbit IgG (cat#A31572), 1:500; Alexa Fluor 488 donkey anti-goat IgG (cat#A11055), 1:500, from Life Technologies) for 1 hour at room temperature and nuclei were stained using DAPI (1:5000).
5
Immunofluorescent Staining of Embryos
6
Immunofluorescence Analysis of Pluripotency Markers in mESCs
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J1 mESCs were treated with DMSO or 3 μM CHIR for 24 h on gelatin-coated 12-well plates. The medium was discarded, and the cells were washed twice with phosphate-buffered saline before being fixed and permeabilized with immunostaining fix solution (Beyotime, Jiangsu, China) for 10 min. After blocking with immunostaining blocking buffer (Beyotime) for 1 h, the cells were incubated with primary antibody in dilution buffer (Beyotime) overnight at 4°C and then with Alexa Fluor 555-secondary antibody (Beyotime) for 2 h at room temperature in the dark. After each step, the cells were washed thrice with immunolstaining wash buffer for 5 min before the next step. DAPI (4′, 6-diamidino-2-phenylin-dole) staining was performed after secondary antibody incubation for 10 min at room temperature. The primary antibodies and dilutions used were as follows: rabbit anti-Klf4 (Abcam, Cambridge, UK; 1:500), rabbit anti-Klf4 (Boster, Wuhan, China; 1:500), mouse anti-Oct4 (Santa Cruz, CA, USA; 1:500), rabbit anti-Cdx2 (Santa Cruz; 1:500), and rabbit anti-Nanog (Cell Signaling Technology, Danvers, MA; 1:500). All reagents not indicated were purchased from the Beyotime Institute of Biotechnology (Beyotime). Immunofluorescence staining was visualized and imaged by a confocal microscope (Nikon, Tokyo, Japan).
7
Pluripotency and Lineage-Specific Markers
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Protein samples were electrophoresed on SDS polyacrylamide gel (10%) and transblotted (MiniTransblot Cell, Bio-Rad, Hercules, CA, USA) onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were probed with rabbit anti-NANOG, anti-SOX17, anti-SOX9, anti-GATA4, anti-GATA6, and anti-FOXA2 antibodies (Aviva Systems Biology), mouse anti-OCT4 (Santa Cruz Biotechnology, San Diego, CA, USA), and rabbit anti-SOX2 antibodies (Abcam, Cambridge, MA, USA) followed by horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies. Mouse anti-beta-actin antibody (Abcam) was used for detecting the loading control. Binding of antibodies was visualized with ECL reagent (Western Lightning Plus-ECL, PerkinElmer Inc, Waltham, MA, USA) and exposing the blots on X-ray films (Amersham Biosciences).
8
Immunofluorescence and Alkaline Phosphatase Staining
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Immunofluorescence (IF) was performed after
fixation of the cultured cells in 4% paraformaldehyde
for 20 minutes followed by permeabilization with
0.2% Triton X-100 (Merk, USA) for 30 minutes.
The cells were subsequently blocked in phosphate-
buffered saline (PBS) supplemented with 10%
secondary antibodies host serum for 1 hour. The
blocked cells were incubated overnight at 4°C with
mouse anti-Oct4 (Santa Cruz, USA, sc5279), mouse
anti-SSEA-1 (R&D, MAB2155) and goat anti-Nanog
(Santa Cruz, USA, sc30329). The cells were washed
three times with PBS and subsequently incubated with
the following secondary antibodies goat anti-mouse
IgG-FITC (Santa Cruz, USA, sc2010), Alexa Fluor
568 goat anti-mouse (Invitrogen, USA, A21043),
and Alexa Fluor 568 donkey anti-goat (Invitrogen,
USA, A11057). The cells were stained with 1 µg/
ml DAPI for 10 minutes in the dark and after three
PBS washes, we used an Olympus fluorescent
microscope (Olympus, Japan) to visualize the cells.
Alkaline phosphatase (ALP) staining was performed
according to the manufacturer’s instructions using an
Alkaline Phosphatase Detection Kit.
fixation of the cultured cells in 4% paraformaldehyde
for 20 minutes followed by permeabilization with
0.2% Triton X-100 (Merk, USA) for 30 minutes.
The cells were subsequently blocked in phosphate-
buffered saline (PBS) supplemented with 10%
secondary antibodies host serum for 1 hour. The
blocked cells were incubated overnight at 4°C with
mouse anti-Oct4 (Santa Cruz, USA, sc5279), mouse
anti-SSEA-1 (R&D, MAB2155) and goat anti-Nanog
(Santa Cruz, USA, sc30329). The cells were washed
three times with PBS and subsequently incubated with
the following secondary antibodies goat anti-mouse
IgG-FITC (Santa Cruz, USA, sc2010), Alexa Fluor
568 goat anti-mouse (Invitrogen, USA, A21043),
and Alexa Fluor 568 donkey anti-goat (Invitrogen,
USA, A11057). The cells were stained with 1 µg/
ml DAPI for 10 minutes in the dark and after three
PBS washes, we used an Olympus fluorescent
microscope (Olympus, Japan) to visualize the cells.
Alkaline phosphatase (ALP) staining was performed
according to the manufacturer’s instructions using an
Alkaline Phosphatase Detection Kit.
9
Detailed Immunofluorescent Staining Protocol
10
Pluripotency and Lineage Marker Analysis
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The following antibodies were used: mouse anti-Oct4 (Santa Cruz), rabbit anti-Nanog54 (link), anti-Sox2 (Calbiochem), rabbit anti-E-cad (extracellular)55 (link), mouse anti-E-cad (intracellular, BD Bioscience), mouse anti-N-cad (BD Bioscience), mouse anti-N-cad (Life Technologies), rat anti-ZO-156 (link), rat anti-pan-Laminin57 , goat anti-T-brachyury (Santa Cruz), goat anti-Sox17 (R&D Systems), rabbit anti-pH3 (Cell Signaling), rabbit anti-H3 (Abcam), mouse anti-β-catenin (BD Bioscience), mouse anti-Ezrin (Cell Signaling) and rabbit anti-pSMAD1-5-8 (Cell Signaling).
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