The generation of the Ruvbl1fl mice was recently described . K14:Cre (MGI: 2177413) expressing the Cre recombinase under the Keratin14 promotor in the epidermis has previously been described . To generate epidermal-specific Ruvbl1-deficient mice, Ruvbl1fl animals and K14:Cre animals were crossed on a C57Bl/6N background. The mice were housed according to standardized specific pathogen-free conditions in the animal facility of the University of Cologne. All matings and experiments were conducted in accordance with European, national and institutional guidelines, as approved by the State Office of North Rhine-Westphalia, Department of Nature, Environment and Consumer Protection (8.87-50.10.31.08.049 and 84-02.04.2013.A152).
For the preparation of the embryonic mice, the pregnant females were sacrificed by cervical dislocation. Tissue was processed by fixation in 4% formaldehyde and embedding in paraffin.
Skin barrier assay
For examination of the skin barrier, intact E18.5 embryos were first dehydrated in increasing amounts of methanol (25%, 50%, 75%, 100%). After rehydration in decreasing methanol solutions (75%, 50%, 25%, 0%), the embryos were stained in 0.0125% toluidine blue for 1 min and finally washed in PBS.
For histological analysis, embryonic tissue was cut in 3-μm-thick sections and stained with hematoxylin and eosin using standard techniques. Paraffin was removed from the embedded tissue by xylene treatment and rehydration in graded ethanol. Sections were then stained, firstly, with Mayer’s hemalum (Sigma-Aldrich) to visualize nuclei in blue, and secondly, with eosin (Carl Roth) to visualize cytoplasmic structures in red.
Embryonic tissue was sliced in 2-μm-thick sections and deparaffinated as described above. Antigen retrieval was achieved using heat-induced epitope retrieval and citrate buffer. For immunohistochemical staining, endogenous peroxidases and unspecific antibody binding sites were blocked by treatment with 3% H2O2 and the ABC Kit according to the manufacturer’s manual (VectorLabs). Primary antibodies were incubated overnight at 4°C in 1% BSA as indicated. After incubation with the biotin-coupled secondary antibody, antibody signals were visualized by developing the tissue in DAB solution (VectorLabs). Nuclear counterstaining was performed using Mayer’s hemalum reagent. For fluorescent staining, tissue was blocked in 5% normal donkey serum (Jackson ImmunoResearch) and 1% bovine serum albumin for 1 h at RT. Primary antibodies were incubated overnight at 4 °C as indicated. Tissue sections were incubated with fluorophore-coupled secondary antibodies and mounted in Prolong Diamond antifade containing DAPI (Invitrogen).
The following antibodies were used: Ki67 (Abcam, 1:500), K14 (Abcam, 1:500), Ruvbl1 (ProteinTech, 1:500), p53 (Leica Biosystems, 1:500), CC3 (Cell Signalling, 1:200), yH2Ax (Cell Signalling, 1:500), ß-cat (Cell Signalling, 1:500), cMyc (Cell Signalling, 1:500).