Cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature and permeabilized with 1% Triton X-100/PBS (Sigma) for 10 min. After blocking with 10% donkey serum in PBS (Jackson ImmunoResearch) for 1 h at room temperature, cells were incubated with primary antibodies overnight at 4 °C, followed by incubation with appropriate fluorescent-conjugated secondary antibodies for 1 h at room temperature the next day. Primary antibodies were as follows: OCT4 (Abcam), SOX2 (Abcam), SSEA4 (Abcam), TFAP2C (Santa Cruz), PRDM14 (Abcam), and NANOS3 (Abcam). Secondary antibodies were as follows: AlexaFluor 488 conjugated donkey anti-rabbit IgG and AlexaFluor 594 conjugated donkey anti-mouse IgG (all Life Technologies). The nuclei were counterstained with DAPI (Thermo Fisher Scientific). The cells were observed with a Zeiss inverted confocal microscope.
Prdm14
Manufactured by Abcam
PRDM14 is a zinc finger protein that functions as a transcriptional regulator. It plays a role in the maintenance of pluripotency in embryonic stem cells.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Nanos3, by Abcam (1 mentions)
Alexa fluor 594 conjugated donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Jackson ImmunoResearch (1 mentions)
Ssea4, by Abcam (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Tfap2c, by Santa Cruz Biotechnology (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Limk1, by Abcam (1 mentions)
P pak1, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Caspase 3, by Abcam (1 mentions)
Hnf4a, by Santa Cruz Biotechnology (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
E cadherin, by Takara Bio (1 mentions)
C myc, by Abcam (1 mentions)
3 protocols using prdm14
1
Immunofluorescence Staining of Stem Cells
2
Western Blot Protein Extraction and Analysis
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Proteins from whole-cell lysates for western blotting were extracted with mammalian protein extraction reagent (cat. no. 78501; Thermo Scientific, Rockford, IL, USA), resolved on SDS–PAGE and transferred to polyvinylidene fluoride (PVDF; cat. no. IPVH00010; EMD Millipore) membrane. The membranes were then blocked with 5% bovine serum albumin (BSA; cat. no. A7906; Sigma-Aldrich, St Louis, MO, USA) in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. The membranes were then incubated with primary antibody for 1 h at room temperature, followed by rinsing of unbound antibody with TBST and incubating the membranes with appropriate horseradish peroxidase-conjugated secondary antibodies. Primary antibodies for Cdc42 (cat. no. ab64533; Abcam, Cambridge, UK), E-cadherin (cat. no. 3195S, Cell Signaling, Danvers, MA, USA), vimentin (cat. no. 5741S; Cell Signaling), p-PAK1 (p-21-activated kinase 1) (cat. no. ab40852; Abcam), LIMK1 (cat. no. ab95186; Abcam), prdm14 (cat. no. ab91587; Abcam), caspase-3 (cat. no. C8487; Sigma-Aldrich) and β-actin (cat. no. A1978; Sigma-Aldrich) were used. Protein expression was detected using SuperSignal West Femto Maximum Sensitivity Substrate (cat. no. 34095; Thermo Scientific) and the membranes were imaged for chemiluminescence using the ImageQuant LAS 4000 Imager (GE Healthcare Life Sciences, Bio-Sciences, Pittsburgh, PA, USA).
3
Immunostaining of Pluripotency and Lineage Markers
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Cells were briefly washed with PBS and fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. Cells were permeabilized for 30 min with 1% BSA and 0.1% Triton X-100 in PBS. Antibody staining was carried out in the same buffer at 4 °C overnight. The slides were subsequently washed three times in PBS with 1% BSA, 0.1% Triton X-100 (5 min each wash), then incubated with secondary antibody for 1 h at room temperature in the dark, washed once for 5 min in 1% BSA, 0.1% Triton X-100 in PBS and twice for 5 min in PBS. The slides were then mounted in Vectashield with DAPI (Vector Laboratories) and imaged using a BioRad Radiance 2100 confocal microscope. Primary antibodies used were: mouse monoclonal Oct4 (BD Biosciences, 1:200), rat monoclonal Nanog (eBioscience, 1:500), goat polyclonal Sox2 (Santa Cruz, 1:200), rabbit polyclonal H3K27me3 (Upstate, 1:500), rat monoclonal E-cadherin (Takara, 1:40), Klf4 (Abcam, 1:300), cMyc (Abcam, 1:200), Esrrb (Abcam, 1:200), Prdm14 (Abcam, 1:200), HNF4a (Santa Cruz, 1:100). All secondary antibodies used were Alexa Fluor highly cross-adsorbed (Molecular Probes).
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