4.5. Immunohistochemistry

Sections (4 µm) of paraffin-embedded intestinal colonic and brain samples from mice were prepared following the standard protocol. Briefly, sections were dewaxed for 13 min at 90 °C with Heat Mediated Antigen Retrieval Solution (“HMARS”, Abcam, Germania, Germany) pH 6.0. Afterward, sections were washed in water for 5 min and peroxidases inhibited by incubation in 3% H₂O₂ for 10 min. Sections were treated with 5% bovine serum albumin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted in phosphate-buffered saline (PBS) for 30 min and incubated with the following primary antibodies: anti-Ki67 (IHC-00375, Bethyl, Montgomery, TX, USA, 1:500), anti-Dcx (ab18723, Abcam, 1:2000, Cambridge, UK), anti-Iba-1 (019-19741, Wako Pure Chemical Industries sections, Osaka, Japan, 1:500) and (ab1872315690, Abcam, 1:2000) were employed for immunostaining of the colon and the brain sections, anti-Gfap (Z0334, Dako, Germania, Germany, 1:500), anti-Sox2 (ab97959, Abcam, 1:500) diluted in PBS, overnight at 4 °C in a moist chamber. They were then washed in PBS, incubated for 1 h at room temperature with the secondary anti-rabbit antibody (Dako North America, Carpinteria, CA, USA) diluted 1:200 in PBS, and washed again. To visualize the antigen, sections were incubated with Vectastein Elite ABC (Vector Laboratories, Inc., Burlingame, CA, USA) for 30 min at room temperature, washed with PBS and incubated with Vector NovaRED Substrate Kit (Vector Laboratories, Inc., Burlingame, CA, USA) for 15 min at room temperature. Finally, samples were stained with H&E and analyzed by light microscopy using the software NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V.; Florence, Italy) at 4×, 10×, 20× and 100× magnification.

For adult neurogenesis, the brain of each mouse was cut sagittally to the midline and sections were collected starting at 500 μm from the midline. Images for quantification were taken using the imaging software NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V.; Florence, Italy). To standardize the counting area, cell quantification was performed on three non-overlapping serially collected sections per mouse, one from each hemisphere, representing the rostral/mid-hippocampus. The number of positive cells in each section was expressed per mm of the SGZ length. NSCs were counted based on criteria including SGZ localization, positive labeling and morphology.

For tumor phenotyping, a subset of brain sections from Ptch1^+/− mice with MB developed during the temporal window of DSS treatments were immunostained for Gfap and Ki67 and specific regions of interest (hippocampus, VIII lobule of cerebellum and tumor tissue) and analyzed by HistoQuest software (Tissue Gnostics, Wien, Austria—for automatic color separation and quantification). All experiments were analyzed in blind.