## 2 Materials and Methods
### Immunolistochemistry
The experiments for Hsp27 were performed using an IHC goat kit (Cell & Tissue Staining Kit, R&D Systems, Inc., Minneapolis, MN, USA, Cat N\({}^{\circ}\) CTS008). Sections, after deparaffinization, were treated at 22 \({}^{\circ}\)C for 5 min with Peroxidase Blocking Reagent (Cell & Tissue Staining Kit, R&D Systems, Inc.) to inhibit endogenous peroxidase activity and after another wash with PBS for 5 min, with a Serum Blocking Reagent D (Cell & Tissue Staining Kit, R&D Systems, Inc.) for 15 min to block non-specific antigenic sites. Since the detection is based on the formation of Avidin-Biotin complex, the sections were treated with Avidin Blocking Reagent (Cell & Tissue Staining Kit, R&D Systems, Inc.) at 22\({}^{\circ}\)C for 15 min. After a wash with PBS, the sections were incubated with Biotin Blocking Reagent for 15\({}^{\prime}\) (Cell & Tissue Staining Kit, R&D Systems, Inc.). After another wash with PBS, the sections were incubated at 22 \({}^{\circ}\)C overnight, with a primary antibody against human Hsp27 (goat anti-Hsp27 polyclonal antibody, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA, cat. N\({}^{\circ}\): sc-1048, dilution 1:150). For all samples, appropriate positive and negative (isotype) controls were run concurrently. So the samples were washed at 22 \({}^{\circ}\)C three times in PBS for 15 min/wash, and then the sections were incubated with Biotinylated Secondary Antibody (Cell & Tissue Staining Kit, R&D Systems, Inc.) for 40 min most often (the length of this incubation time varies depending on the thickness of the section). After that, the slides were washed three times in PBS for 15 min/wash. The high detection sensitivity of this method is obtained by using premium quality biotinylated secondary antibodies and High Sensitivity Streptavidin-conjugated HRP (HSS-HRP). HSS is a chemical analog of Streptavidin that interacts only with Biotin bound to secondary antibodies. The sections were incubated at 22 \({}^{\circ}\)C with HSS-HRP (Vial B, Cell & Tissue Staining Kit, R&D Systems, Inc.) for 30 min. For all samples, the visualization is based on enzymatic conversion of chromogenic substrates 3,3\({}^{\prime}\) Diaminobenzidine (DAB) into a colored precipitate (brown), by horseradish peroxidase (HRP) at the sites of antigen localization, after two subsequent washing with PBS for 2 min/wash, the slides were incubated in the dark for 5 min with the DAB chromogen (200 \(\mu\)L of DAB chromogen solution were required to cover tissue section on a single slide). Two drops of DAB Chromogen were added to 2 mL of DAB Chromogen Buffer (Cell & Tissue Staining Kit, R&D Systems, Inc.). The nuclei were counterstaineded with hematoxylin (Hematoxylin aqueous formula, REF 05-06012/LN. Cat. S2020, Bio-Optica, Milano, Italy). Finally, the slides were mounted for observation with coverslips using a permanent mounting medium (Vecta Mount, H-5000, Vector Laboratories, Inc. Burlingame, CA, USA). The observation of the sections was carried out with an optical microscope (Leica DM 5000 B, Leica Microsystems Srl, Buccinasco (MI), Italy) connected to a digital camera (Leica DC 300F). Two independent observers (F.C, and F.R) examined all specimens on two separate occasions and performed a quantitative analysis to quantify the percentage of epithelial cells positive for Hsp27, Hsp60, Hsp70, and Hsp90. All the observations were done at a magnification of 400\(\times\) and the percentage of positive cells was calculated in a high-power field (HPF) and repeated for 10 HFP. The immunopositivity evaluation is expressed as average percentage of all immuno-quantifications performed in each case for each Hsp. Statistical analyses were carried out using the GraphPad Prism 4.0 package (GraphPad Inc., San Diego, CA, USA). One-way ANOVA analysis of variance with Bonferroni post-hoc multiple comparisons was used to detect significant statistical differences. All data are reported as the means \(\pm\) SD, and the level of statistical significance was set at \(p\leq 0.05\).