Deparaffinization
Deparaffinize the tissue sections from adult murine skin by submerging the sections in the following alcohol row: xylol for 5 minutes, fresh xylol in a different container for 5 minutes, isopropanol for 5 minutes, 96% ethanol for 5 minutes, 75% ethanol for 5minutes, 50% ethanol for 5 minutes, and H2O for 5 minutes.
Antigen retrieval
Retrieve the epitopes by boiling the sections in Dako Antigen Retrieval Agent pH9 (Dako) for 20 minutes inside a boiling cuvette in a pressure cooker.
Allow slides to cool down within the retrieval solution for 1h at room temperature and then wash twice with PBS++.
Epitope dephosphorylation
Delimit the tissue sections with a fat pen (Dako pen).
Incubate tissue sections with 25U Antarctic Phosphatase in AP buffer, or with AP buffer only (vehicle control) for 2 hours at 37°C in a humidified chamber.
Blocking
Block unspecific binding sites with 5% BSA in PBS++ for 1h at room temperature in a humidified chamber. Ensure that the blocking solution covers the entire tissue section.
Proceed with standard immunostaining protocols.