Precellys system

All animal procedures were approved by the Animal Care and Use Committee of the Medical University of Vienna (66.016/0014-II/3b/2011), and all these procedures were conducted according to the guidelines established by this committee. Fertilized chicken eggs were incubated according to published protocols (Eresheim et al. 2014 (link)). Hatched chickens were obtained by Schropper GmbH, Gloggnitz, Austria. Chickens were decapitated at embryonal development day 18 or on the first day after hatching. Tissue samples for protein analysis were frozen immediately at −20 °C until further processing. Proteins were extracted with Laemmli extraction buffer containing 2% SDS by homogenization with the Precellys system (VWR International, Radnor, PA). Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Fisher Scientific). For analyses of RNA, samples from different chicken tissues were cut into small pieces and kept in TriFast (VWR International) at 4 °C overnight. RNA was purified using the Precellys system (VWR International, Radnor, PA) and TriFast according to the manufacturers’ instructions.

Proteins from cornea and epidermis samples were homogenized with the Precellys system (VWR International, Radnor, PA) in Laemmli extraction buffer containing 2% SDS. Protein concentrations were determined with the Micro BCA protein assay kit (Thermo Fisher Scientific). Thirty µg of total protein per lane were electrophoresed through an ExcelGel SDS 8–18% polyacrylamide gradient gel (GE Healthcare Life Sciences, Chicago) and blotted onto a nitrocellulose membrane. The membrane was incubated with guinea pig anti-K24 (dilution 1:1000) overnight at 4 °C. After washing, the membrane was incubated for 1 h at room temperature with goat anti-guinea pig IgG (Abnova, AB10611, 1:10000) coupled to horseradish peroxidase. Bands were visualized using the enhanced chemiluminescence system (SuperSignal West Dura Extended Duration Substrate, Thermo Fisher Scientific). The membrane was reincubated with monoclonal mouse anti-K14 (Abcam, ab7800, 0.33 µg/ml) and sheep anti-mouse IgG (GE Healthcare, NXA931V, 1:10000) coupled to horseradish peroxidase as a secondary antibody.