A5597

For each FFPE tumour sample, a 3 µm–thick section was examined histologically after haematoxylin and eosin staining to assess the percentage of tumour cells. Adjacent 5 μm–thick sections were then macro-dissected to enrich in tumour cells (>50%) and used for lysate preparation. The number of sections was adjusted to the tumour area (ideally three sections for a tumour with an area of 100 mm² after macro-dissection). Sections were placed in a 1.5 ml microcentrifuge tube containing 1 ml of xylene substitute (#A5597, Sigma, Ile d’Abau, France). Paraffin was dissolved by incubation at room temperature (RT) for 5 min, and then removed by centrifugation. Pellets were washed to remove residual contaminants (absolute ethanol twice, then 95% ethanol), collected by centrifugation (12,000 RCF) and resuspended in 1X Tris/EDTA pH 9 (Target Retrieval Solution #S2367, Dako). Samples were then heated at 95 °C in a Thermomixer (Eppendorf, Hamburg, Germany) for 45 min, centrifuged to remove the Tris/EDTA buffer, and resuspended in 250 µl of Lysis Buffer (LB4 #64KL4FDF, Cisbio, Codolet, France). Finally, ice-cold samples were lysed by sonication (80 W for 15 s) using a Vibra-Cell™ sonicator (Sonics & Materials, Newtown, CT, USA). Lysates were clarified by centrifugation (10,000 RCF) at 4 °C for 5 min and then transferred to the wells of a microtiter plate.

Following the modeling of rat in groups aforementioned, skin flap was obtained and fixed immediately by 4% formaldehyde from rats after the perfusion of PBS via circulatory system. Next, skin flap were cut into piece in order to embed into paraffin after 24 h of fixing. Briefly, the pieces of flap tissue were dewaxed by xylene substitute (A5597, Sigma-Aldrich, Missouri, USA) and then stained with hematoxylin (#14166, CST, MA, USA) and eosin (E4009, Sigma-Aldrich, Missouri, USA), following the procedures of HE staining provided by manufacturer of solutions purchased. The structure and cells in the flap tissue were further observed and recorded.