z stack confocal images  (Olympus)

Journal: Development (Cambridge, England)

Article Title: Kindlins regulate integrin- and growth factor-dependent ureteric bud formation

doi: 10.1242/dev.205044

Figure Lengend Snippet: Y795A and TTAA CD cells have defective integrin-dependent functions. (A) Control, Y795A and TTAA mutant CD cells were placed in 3D collagen I/Matrigel gels for 7 days, stained with rhodamine-phalloidin and visualized by confocal microscopy. (B) CD cell populations were allowed to adhere to Matrigel at the indicated concentrations for 1 h. Percentage adhesion is demonstrated on the y -axis and the concentration of Matrigel is on the x -axis ( n =3 experiments). Significance (* P <0.05) is indicated between control and Y795A or TTAA at the indicated concentration (unpaired t -test). (C) CD cells were plated on transwells coated with Matrigel and the number of cells that had migrated after 4 h was counted and expressed as cells per high powered field (HPF). n ≥6 HPFs pooled from three experiments. (D) CD cells were plated and allowed to spread for 1 h on Matrigel, stained with rhodamine-phalloidin (F-actin) and visualized. (E) CD cells were plated on Matrigel after which they were stained with rhodamine phalloidin and antibodies directed against phospho-paxillin (P-Pax, green), and visualized with confocal microscopy. Images are representative of n =3 repeat experiments. (F) Quantification of spreading area per cell of at least 20 cells per group. (G) Quantification of focal adhesion (FA) number per cell of at least 10 cells per group. (H) CD cell proliferation, as assessed by BrdU incorporation, 1 h after plating on Matrigel colorimetrically (OD 450 nm). The averages of three experiments are shown. (I) CD cells were grown to confluence as monolayers on transwells, and stained for F-actin (rhodamine phalloidin green), ZO-1 (tight junctions, magenta) and nuclei (DAPI, blue). Shown are xz -orthogonal projections sliced at the yellow dotted line of the z -stack cross-section. Single-channel images of ZO-1 and F-actin are shown as inverted grayscale images. (J) Quantification of polarity by polarization index (ratio of apical over total ZO-1 immunofluorescence). Averages of four monolayers per group are shown. Images are representative of three samples per group from 3 independent replicates. * P <0.05; ns, not significant (ANOVA with post-hoc Tukey's test). Data are mean±s.d. Scale bars: 40 µm (A); 20 µm (D,I); 10 µm (E).

Article Snippet: 3D reconstructions of z -stack confocal images was performed in IMARIS (Bitplane).

Techniques: Control, Mutagenesis, Staining, Confocal Microscopy, Concentration Assay, BrdU Incorporation Assay, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: Kindlins regulate integrin- and growth factor-dependent ureteric bud formation

doi: 10.1242/dev.205044

Figure Lengend Snippet: Kindlin 1/2 double-null CD cells are unable to adhere, migrate or spread. (A) Immunoblotting of control and K1K2 −/− CD cell total lysates for kindlin 1 and kindlin 2 with actin as the loading control. (B) Control and kindlin 1/2 double-null CD cells (K1K2 −/− ) were placed in 3D collagen I/Matrigel gels for 7 days, stained with rhodamine-phalloidin and visualized by confocal microscopy to assess tubulogenesis. (C) CD cell populations were allowed to adhere to Matrigel at the indicated concentrations for 1 h. n =3 experiments. * P <0.05 between control and K1K2 −/− CD cells at the indicated concentration (unpaired t -test). (D) CD cells were plated on transwells coated with Matrigel, and migration, measured as cells per high powered field (HPF), was evaluated after 4 h. n ≥6 HPFs pooled from three experiments. (E) CD cells were allowed to spread for 1 h on Matrigel and stained with rhodamine-phalloidin (F-actin). (F) After spreading, rhodamine-stained (magenta) cells were co-labeled for the focal-adhesion protein phospho-paxillin (P-Pax, green) and visualized with confocal microscopy. Data are representative of n =3 repeat experiments. (G) Quantification of spreading area per cell of at least 20 cells per group. (H) Quantification of focal adhesions (FAs) per cell of at least 10 cells per group. (I) CD cell proliferation, as colorimetrically (OD 450 nm) assessed by BrdU incorporation 1 h after plating on Matrigel. Averages of three experiments are shown. (J) Transwell polarity assay of CD cell monolayers grown to confluence over 24 h in transwell inserts and visualized with confocal microscopy of phalloidin (F-actin, green), ZO-1 (tight junctions, magenta) and nuclei (DAPI, blue). Shown are xz -orthogonal projections sliced at the yellow dotted line of the z -stack cross-section. Single-channel images of ZO-1 and F-actin are shown as inverted grayscale images. (K) Quantification of polarity by polarization index (ratio of apical over total ZO-1 immunofluorescence). Averages of four monolayers per group are shown. (L) Immunoblotting of CD cell total lysates for E-cadherin (Ecad) and ZO-1 with GAPDH as the loading control. Representative of three samples per group from independent replicates. The control data are reproduced from Fig. 3 as these experiments were performed simultaneously. * P <0.05; ns, not significant (unpaired t -test). Data are mean±s.d. Scale bars: 40 µm (B); 20 µm (E,J); 10 µm (F).

Article Snippet: 3D reconstructions of z -stack confocal images was performed in IMARIS (Bitplane).

Techniques: Western Blot, Control, Staining, Confocal Microscopy, Concentration Assay, Migration, Labeling, BrdU Incorporation Assay, Immunofluorescence