Immunohistochemical staining was performed as follows: 3-μm-thick sections were prepared from formalin-fixed paraffin-embedded TMA tissue blocks and dried in a 37 °C oven overnight. The sections were placed in a Bond Max Automated Immunohistochemistry Vision Biosystem (Leica Microsystems GmbH, Wetzlar, Germany) according to the following protocol. First, tissues were deparaffinized and pre-treated with the Epitope Retrieval Solution 2 (pH9) at 100 °C for 20 min. After washing steps, peroxidase blocking was carried out for 10 min using the Bond Polymer Refine Detection Kit DC9800 (Leica Microsystems GmbH). Tissues were again washed and then incubated for 30 min with the primary antibody diluted (1:50 anti-Rab35, Abcam ab 152138) in Bond Primary Antibody Diluent (AR9352). Subsequently, tissues were incubated with post primary and polymer for a total of 16 min and developed with DAB-Chromogen for 10 min and counterstained with haematoxilin for 5 min.
Human samples were collected with inform consent between 1995 and 2010, unfortunately, without recording their clinico-pathological features, thus impeding the analysis of RAB35 expression levels with relevant tumour properties.
Human samples were collected with inform consent between 1995 and 2010, unfortunately, without recording their clinico-pathological features, thus impeding the analysis of RAB35 expression levels with relevant tumour properties.