inverted widefield microscope ti eclipse  (Nikon)

Journal: Development (Cambridge, England)

Article Title: In vitro modelling of anterior primitive streak patterning with human pluripotent stem cells identifies the path to notochord progenitors

doi: 10.1242/dev.202983

Figure Lengend Snippet: Timely TGFβ inhibition directs NotoPs on micropatterns. (A) FISH staining for NODAL , CHRD and NOTO in 500 µm micropattern colonies induced with 2 µM CHIR and 20 ng/ml FGF2 and fixed at 48 h post-induction. Timed treatment with the NODAL or BMP inhibitors is indicated. (B) Box plots of average signal intensities in each colony (left) and heatmaps of binned signal intensities along the radial distance (right). ** P <0.05; *** P <0.001 (unpaired t -tests). Box limits indicate the 1st and 3rd quartiles of the distribution, horizontal bars indicate the median and whiskers show 1.5 times the interquartile range (delimited by the box). Points outside this range are considered outliers (diamonds). (C) Widefield images of micropatterned colonies (NAS2) stained by FISH at 48 h with SL added at 24 h. Yellow arrows indicate a 100 µm domain from the edge. (D) z -stack maximum projections of colonies stained 2 days post-CHIR/FGF induction and SB and LDN (SL) treatment as indicated. Insets show boxed areas at higher magnification. (E) z -stack maximum projections of colonies grown for 3 days in 2 µM CHIR and 20 ng/ml FGF-containing medium and an additional 3 days in unsupplemented N2B27 with SL treatment as indicated. (F) Confocal sagittal plane of a whole-mount immunostained early bud mouse embryo. Notice the SOX9 expression in the crown cells of the node and the nascent notochord. Scale bars: 100 µm. A.U., arbitrary unit.

Article Snippet: Micropatterned colonies were imaged on an inverted widefield microscope (Nikon Eclipse Ti) with a long distance 10× lens to image entire wells (8 mm×8 mm).

Techniques: Inhibition, Staining, Expressing

Journal: Development (Cambridge, England)

Article Title: In vitro modelling of anterior primitive streak patterning with human pluripotent stem cells identifies the path to notochord progenitors

doi: 10.1242/dev.202983

Figure Lengend Snippet: CHIR dosage defines downstream dynamics of NODAL signalling. (A) Diagrams illustrating the position of the image crops shown in B and D-G (left) and the organisation of the bins shown in heatmaps (middle). (B-G) Time course analysis of WNT and NODAL signalling in 500 µm colonies across CHIR concentrations. B and E show immunofluorescence analysis of nuclear LEF1 and nuclear SMAD2/3 levels, respectively. Images show selected z -slices across a confocal z -stack. D, F and G show FISH analysis of NODAL , LEFTY2 and CER1 transcript levels. Images are 2D widefield images. For B,D-G, the radial profiles of signal intensities over time is shown as heatmaps. (C) Temporal profiles of protein (LEF1 and SMAD2/3) and transcript (NODAL, LEFTY2 and CER1) levels averaged across entire colonies. Lines indicate the mean average expression across colonies, and the shaded area indicates the 95% confidence interval. The figure is representative of two independent experiments. A.U., arbitrary unit.

Article Snippet: Micropatterned colonies were imaged on an inverted widefield microscope (Nikon Eclipse Ti) with a long distance 10× lens to image entire wells (8 mm×8 mm).

Techniques: Immunofluorescence, Expressing