probe hybridization buffer phb  (Molecular Instruments)

Journal: Development (Cambridge, England)

Article Title: Early autonomous patterning of the anteroposterior axis in gastruloids

doi: 10.1242/dev.202171

Figure Lengend Snippet: Autonomous AP axis formation in gastruloids is demarcated by T polarization and accompanied by variable cell behaviours. (A) Gastruloids were generated from T::GFP mESCs as previously described , including a pulse of CHIR99 from 48 to 72 hpa. T symmetry breaking occurs by 48 hpa. Scale bar: 200 µm. Right panel displays T fluorescence intensity of the same n =23 gastruloids at 24, 48 and 72 hpa, plotted along the normalized AP axis length. Central lines indicate the mean intensity values of the included samples; surrounding shades mark the standard deviation. (B) Averaged kymograph of T expression along the AP axis of n =13 gastruloids, live imaged from 24 to 48 hpa, illustrates progression of T polarization over developmental time. (C-E) Hybridization chain reaction (HCR) in situ stainings for T, Foxa2 (anterior mesendoderm) and Sox2 (pluripotent and neural progenitors) in gastruloids at 24, 48 and 72 hpa, showing the arrangement of germ layer primordia pre- and post-T polarization. Scale bars: 100 µm. Right hand panels show quantifications of T and Sox2 fluorescence along the gastruloid AP axis (defined by localization of T expression) based on sum intensity projections of the HCR data for each timepoint. Data are mean±s.d. (F) Representative images of live imaging of gastruloids from T::mESCs, captured via LSFM showing the T polarization process and associated cellular dynamics. Aggregate shape was inferred via SIR-DNA counterstains. Scale bar: 100 µm. (G) Examples of cell behaviours displayed across LSFM datasets by T + cells during aggregate-wide polarization. White arrowheads indicate cell protrusions; yellow arrowheads indicate cell shape change. Scale bars: 10 µm. The contrasts of the fluorescent channels have been adjusted for display purposes.

Article Snippet: Samples were then transferred to a 1.5 ml Eppendorf tube and 500 µl probe hybridization buffer (PHB, Molecular Instruments, HCR v3.0) was applied for 30 min at 37°C.

Techniques: Generated, Fluorescence, Standard Deviation, Expressing, Hybridization, In Situ, Imaging

Journal: Development (Cambridge, England)

Article Title: Early autonomous patterning of the anteroposterior axis in gastruloids

doi: 10.1242/dev.202171

Figure Lengend Snippet: Spatial AP axial patterning in gastruloids before external Wnt stimulation. (A) Expression of marker genes for anterior (asterisk indicates anterior only in gastruloids, due to absence of cranial patterning), trunk and posterior tissues on the integrated (24, 48 and 72 hpa) UMAP that is filtered for cells from only 48 and 72 hpa. Segregation of expression clusters occurs already at 48 hpa, before external CHIR99 application. Insets in the 72 hpa UMAPs display normalized T (as a reference) and AP marker gene expression plotted along inferred (developmental) pseudotime based on the integrated scRNA-dataset. (B,C) Hybridization chain reaction (HCR) in situ for key AP axial and germ layer marker genes T (posterior, early mesoderm), Aldh1a2 (trunk tissues) and Eomes (anterior mesendoderm) at 48 (B) and 72 (C) hpa. Spatial segregation of expression patterns is initiated by 48 hpa. Scale bars: 100 µm. The contrasts of the fluorescent channels have been adjusted for display purposes.

Article Snippet: Samples were then transferred to a 1.5 ml Eppendorf tube and 500 µl probe hybridization buffer (PHB, Molecular Instruments, HCR v3.0) was applied for 30 min at 37°C.

Techniques: Expressing, Marker, Hybridization, In Situ

Journal: Development (Cambridge, England)

Article Title: Early autonomous patterning of the anteroposterior axis in gastruloids

doi: 10.1242/dev.202171

Figure Lengend Snippet: T polarization and AP axial patterning are robust to changes in aggregate size. (A,B) Representative images (A) and quantitative analyses (B) of T expression along the normalized AP axis of gastruloids grown from different initial cell numbers (N) demonstrates the reproducibility of T symmetry breaking and scaling of relative T domain size between 50 and 1000 starting cells. (C,D) Hybridization chain reaction (HCR) in situ of gastruloids from different initial cell numbers at 72 hpa for key germ layer markers and AP axial patterning genes T, Foxa2 and Sox2 (C), as well as T, Aldh1a2 and Eomes (D). Scale bars: 100 µm. The contrasts of the fluorescent channels have been adjusted for display purposes. (E) Quantification of AP patterning gene expression along the gastruloid AP axis (defined by localization of T) based on sum intensity projections of HCR data from D. The segregation of expression domains is robust, except for N i = 1000. Data are mean±s.d.

Article Snippet: Samples were then transferred to a 1.5 ml Eppendorf tube and 500 µl probe hybridization buffer (PHB, Molecular Instruments, HCR v3.0) was applied for 30 min at 37°C.

Techniques: Expressing, Hybridization, In Situ