2.3. Immunofluorescence (IF) Analysis
Cell array slices were deparaffinized and rehydrated by incubating in solutions with decreasing alcohol content. Antigen retrieval was conducted by boiling the sample slides in citrate buffer 0.01 M pH 6. Both coverslip and CMA slices were incubated in 0.1 M glycine for 10 min at RT and then in blocking solution composed by 5% donkey serum, 0.6% Triton in PBS for 30 min at RT. Samples were immunostained at 4 °C overnight in blocking solution with primary antibodies anti-Nestin (1:50; Millipore, Milan, Italy), anti-sox2 (1:300; Millipore), anti-β3-tubulin (1:100; Sigma) and anti-sel1L (5 μg/mL) [16]. For the immunofluorescence characterization, samples were incubated with appropriate secondary antibodies (Rhodamine-Red antimouse IgM and anti-rabbit IgG, Alexa Fluor 488 anti-mouse IgG, (Jackson ImmunoResearch, distributed by Li StarFish, Milan, Italy) and the nuclei were counterstained with Hoechst 33258. The samples were mounted with GelMount aqueous mounting medium (Sigma). The images were acquired using a Leica DMI4000B inverted microscope linked to a DFC360FX or to a DFC280 cameras (Leica Microsystems).