Computational protocol: Sox17 is indispensable for acquisition and maintenance of arterial identity

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Protocol publication

[…] For cell immunofluorescence, confluent endothelial cells were fixed with 4% paraformaldehyde (PFA) in PBS, followed by permeabilization with 0.5% Triton X-100 for 5 min. The blocking (1 h), primary (2 h) and secondary (1 h) antibodies were in PBS with 2% BSA. The nuclei were visualized using DAPI.Retina immunostaining was carried out with littermates processed simultaneously under the same conditions. To analyse and quantify the retina vascular phenotype, the eyes (P5, P9 and P12) were fixed in 2% PFA for 2 h at 4 °C and dissected. After blocking/permeabilization in 1% BSA, 5% donkey serum and 0.5% Triton-X100 in PBS overnight, the retinas were washed in Pblec Buffer (1% Triton X-100, 1 mM CaCl2, 1 mM MgCl2 and 0.1 mM MnCl2 in PBS, pH 6.8) and incubated overnight at 4 °C in Pblec buffer containing biotinylated isolectin B4 (IB4) and the primary antibodies. Then the retinas were incubated with fluorophore-conjugated antibodies and with Alexa Fluor 488/555/647 streptavidin (Invitrogen), and mounted with ProLong Gold (Invitrogen).For whole-mount immunostaining, embryos were dissected in PBS and fixed in 4% PFA overnight at 4 °C. After blocking/permeabilization for 3 h in PBS containing 0.3%TX-100 and 5% donkey serum, embryos were incubated overnight at 4 °C with the primary antibodies followed by the species-specific Alexa Fluor-coupled secondary antibodies.Cryostat sections (8–12 μm thick) were cut using a cryotome (Leica), mounted on Superfrost glass slides and dried. Sections were permeabilized for 5 min in PBS containing 0.5% TX-100, blocked in 2% BSA, 5% donkey serum and 0.05% TX-100 in PBS and incubated overnight at 4 °C with the primary antibodies. Secondary detection was performed with Alexa Fluor-coupled secondary antibodies for 3 h at room temperature. Speciments were mounted with Vectashield (Vector Labs).Cerebella (P5) were dissected, fixed and embedded in 3% low melting point agarose and 100 μm sections were cut on vibratome and immunostained.After anaesthesia, mice were perfused for 2 min with fixative (1% PFA in PBS, pH 7.4) from a cannula inserted through the left ventricle into the aorta.The aorta, diaphragm, urinary bladder and intestine were fixed in 1% PFA at pH 7.5 for 1 h, antibodies were diluted in 5% donkey serum in PBS containing 0.3% Triton-X100. The stained tissues were mounted with Vectashield (Vector Labs).Confocal microscopy was performed with Leica TCS SP2 and SP5 confocal microscopes. ImageJ (NIH) was used for the data analysis.The figures were assembled using Adobe Photoshop and Adobe Illustrator. The only adjustments used in the preparation of the figures were for brightness and contrast. For comparison purposes, different sample images of the same antigen were acquired under constant acquisition settings. […]

Pipeline specifications

Software tools ImageJ, Adobe Illustrator
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus
Diseases Arteriovenous Malformations