Phospho h3 ser 10

For immunofluorescence staining in skin, pancreas, mammary gland and intestine, sections were thawed at room temperature for 15 min and encircled with DAKO hydrophobic pen. Then, they were washed 3x for 5 min with PBS. Antigen retrieval was performed by adding pre-warmed citrate buffer pH = 6.0 to the samples and incubating them at 85°C for 30 min. Samples were washed 3x for 5 min with PBS, then incubated in blocking solution (10% horse serum, 0.5% Triton X-100 in PBS) for 1h at room temperature. Primary antibodies were diluted in staining solution (5% horse serum, 0.5% Triton X-100 in PBS) and added to the samples over night at 4°C. Next day, samples were washed 3x for 5min with PBS and incubated with secondary antibodies (1:1000) and Hoechst (Sigma, 1mg/ml stock, 1:1000) diluted in staining solution for 2hrs at room temperature. After washing 3x for 5min with PBS, samples were mounted with Mowiol and stored at 4°C until they were imaged at a Zeiss LSM800. Primary antibodies: Keratin 8 (Abcam, 1:100), Keratin 14 (BioLegend, 1:500), beta-Catenin (Cell Signaling, 1:100), phospho-H3(Ser10) (Cell Signaling, 1:800). Secondary antibody: donkey anti-rabbit Alexa647 (Molecular Probes). Mounted brain sections were washed 3x for 5 min in PBS, DAPI stained (1:20’000) for 10 min and then embedded in mounting medium containing 1,4-diazabicyclooctane (DABCO; Roth) and Mowiol (Roth).