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 separate 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++.
Blocking
Delimit the tissue sections with a fat pen (Dako pen).
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.
Primary Antibodies
Incubate tissue sections with primary antibodies diluted in antibody buffer overnight at 4°C in a humidified chamber. Verify that the antibody solution covers the entire tissue section. Use pMLC2 Ser19 (CST #3675) at 1:200 dilution and ppMLC2 Thr18/ Ser19 (CST #3674) at 1:100 dilution.
Secondary Antibodies
Wash the tissue sections 2x with PBS++ for 5 minutes each.
Incubate tissue sections with secondary antibodies and DAPI (2µg/ml) for 1h at room temperature in a humidified chamber in the dark.
Mounting
Wash the tissue sections 2x with PBS++ for 5 minutes each.
Briefly wash the tissue sections 1x with H2O.
Mount tissue sections in Mowiol. Ensure that the Mowiol solution covers the entire tissue section. Remove any air bubbles.
Cover with coverslip.
Let the Mowiol solidify for 24 hours (store dry in the dark at room temperature).
Seal slides with quick-dry nail polish.
Store samples in the cold and dark until further analysis.