primary anti nanog  (Cell Signaling Technology Inc)

Journal: Scientific reports

Article Title: Topological data analysis of pattern formation of human induced pluripotent stem cell colonies.

doi: 10.1038/s41598-025-90592-1

Figure Lengend Snippet: Fig. 2. Artificial induction of exogenous GATA6-HA occurs within the context of endogenous GATA6 expression. (a) Gene circuit for chemical induction of GATA6-HA expression. The pan-GATA6 antibody can detect both induced GATA6-HA and endogenous GATA6 (i.e. the total amount of GATA6), whereas the HA antibody can only detect induced GATA6-HA (i.e. a subset produced via induction of the total amount of GATA6). (b) Representative immunofluorescence images of NANOG and pan-GATA6 or HA at 0 and 25 ng/ml Dox concentrations (scale bar, 440µm). Using Volocity, we applied the gamma changes (gamma of 1.5) after brightness enhancement on all stitched large images used in the figure for better contrast for representation (see Methods). (c) Quantification of segmented images by each channel from (b). Green (NANOG) and red (pan-GATA6 or HA) fluorescent intensities are normalized to the corresponding nuclear Hoechst value in blue. The threshold for the signal in the green channel is given by the green dotted line, and the red dotted line indicates the threshold for the signal in the red channel. The four cell types based on these thresholds are given by the labels R+G−, R+G+, R−G+, and R−G− of the corresponding quadrant.

Article Snippet: Primary antibodies are Nanog (Cell Signaling Technology 4893S, 1:2000), Gata6 (Abcam ab22600, 1:200, R&D Systems AF1700, which was only used in co-staining, 1:200), and HA (Novus Biologicals NB600-363R, 1:200).

Techniques: Expressing, Produced, Immunofluorescence

Journal: Frontiers in immunology

Article Title: A novel iPSC-based model of ICF syndrome subtype 2 recapitulates the molecular phenotype of ZBTB24 deficiency.

doi: 10.3389/fimmu.2024.1419748

Figure Lengend Snippet: FIGURE 1 Molecular characterization of pYM-iPSCs. (A) Expression of pluripotency markers hTERT, LIN28, SOX2, DNMT3B, OCT3/4, NANOG and KLF4 transcripts measured by RT-PCR in a representative clone of pYM-iPSCs (C6); (B) Expression of OCT3/4, NANOG and DNMT3B pluripotency markers measured by immunofluorescence in the pYM C6 clone. DAPI, nuclear staining. Bar: 100mm; (C) Dot plot showing the expression of the pluripotency marker SSEA4 in the pYM C6 clone compared to the negative control (CTRL-). PE-CF594-A, conjugated secondary antibody; (D) Karyotype analysis demonstrating chromosomal integrity of the pYM C6 clone; (E) Immunostaining of markers representing the three embryonic germ layers, GATA4 (endoderm), bIII-tubulin (ectoderm) and MF20 (mesoderm) expressed in the in vitro differentiated C6 clone. Bar: 100 mm; (F) ZBTB24, CDCA7, CDC40 and OSTC transcripts level in control (C14 and C22) and pYM (C6 and B9) iPSC clones measured by qPCR. The relative expression compared to the expression of GAPDH is shown for each gene. qPCR data are presented as mean ± SD from independent triplicates. Statistical analyses were performed using a one-side two-sample Student’s t-test compared to control: (*) P-value < 0.05, (***) P-value < 0.001.

Article Snippet: Primary antibodies against NANOG (SCTB, sc-293121), DNMT3B (Diagenode, pAb-076-005), OCT3/4 (SCTB, sc-5279), GATA4 (SCBT, SC25310), bIII-tubulin (Sigma-Aldrich, T8660), MF20 (DSHB, RRID: AB_2147781) were incubated overnight at 4° C. The cells were then incubated with secondary antibodies: AlexaFluor 594 conjugated goat anti-mouse IgG, AlexaFluor 594 Frontiers in Immunology 03 conjugated donkey anti-rabbit IgG (ThermoFisher).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Marker, Negative Control, Immunostaining, In Vitro, Control, Clone Assay