*4.2. Immunohistochemistry (IHC)*

The immunohistochemical staining for the identification of Antigens has been achieved using BenchMark XT automatic system (Ventana Medical System, Inc., Tucson, AZ, USA), according to protocols that needed standardization for different types of antibodies. The sections obtained from the selected paraffin-embedded blocks were dewaxed in xylene, rehydrated in ethanol, and rinsed in distillated water. The antigen retrieval was made by using the Heat-Induced Epitope Retrieval (HIER) procedure, with an antigen retrieval solution of pH 9 using CC1 solution (Ventana Medical System, Tucson, AZ, USA), consisting of a combination of ethylenediaminetetraacetic and boric acid diluted in Tris buffer for 30—60 minutes. After the endogenous peroxidase blocking with 3% hydrogen peroxide and treatment with normal goat serum 10%, used to block the non-specific protein bonds, the sections were incubated with the primary antibody BMI-1 (clone F6/ABCAM, 1/50 dilution, Abcam, Cambridge, MA, USA). Consequently, the incubation with Ultra-Vision Quanto Detection System Horseradish peroxidase (HRP) (Igs; Ventana Medical Systems) has been performed. Antigen-antibody reaction has been visualized using 3,30 - Diaminobenzidine as a chromogen (UltraView, Ventana Medical Systems, Tucson, AZ, USA). The counterstaining of the sections was done with Mayer's Hematoxylin. After counterstaining, the slides have been washed with liquid soap in order to eliminate the oily film, they have been rinsed with taping water and have been also bathed twice in distilled water. Negative controls have been used for results interpretation, in which primary antibodies have been skipped and replaced with distilled water and positive controls have been considered as endothelial cells and stromal fibroblasts immunostaining.
