Altra 20 digital camera

Paraffin-embedded tissue sections (4 μm) were deparaffinized, subjected to antigen retrieval in hot 10 mM sodium citrate buffer, and then blocked with BSA solution for 1 h at RT. They were incubated with primary antibodies overnight at 4 °C, washed with PBS, incubated with secondary antibodies for 30 min at RT, and developed with DAB reagent (Dako). Sections were counterstained with hematoxylin, dehydrated, and then coverslips were mounted with DPX (Sigma) and slides observed and photographed under the light microscope CX40 Olympus, equipped with the Altra-20 digital camera^{51 (link)}.

Wt or transfected cells were plated (2 × 10⁴ cells per plate) and cultured for 15 days. Colonies were then fixed in 4% paraformaldehyde, stained with crystal violet (0,1% in 20% methanol) and scored.
Colonies were photographed under the CKX41 phase-contrast microscope Olympus (Tokyo, Japan) equipped with the Altra-20 digital camera, and with the AnalySIS^® getIT imaging acquisition software (Olympus). For soft agar assay, wt or transfected cells were suspended in DMEM medium with 10% FBS and 0.3% agarose (SeaKem^® ME by Lonza, Basel, Switzerland), and plated on top of a 0.6% agarose layer at the concentration of 2 × 10⁴ cells per p60 plate. Growing colonies were scored after 20 days and photographed under phase-contrast microscope.

Neurospheres formation assay was performed in serum free medium containing half mixture of F12 and DMEM Low Glucose, supplemented with EGF 20 ng/mL, bFGF 40 ng/mL, 2% B27 (Gibco, ThermoFisher Scientific) and 1% l-glutamine/penicillin–streptomycin. Cells were seeded in suspension culture dishes or in standing culture flasks. Starting from the third or fourth day following the cells seeding in serum-free medium, spheres were observed and photographed under the CKX41 phase-contrast microscope Olympus (Tokyo, Japan) equipped with the Altra-20 digital camera, and with the AnalySIS-getIT imaging acquisition software (Olympus). Neurospheres were collected and isolated from single cells for Western blot analysis, by filtering the culture medium with nylon filters (41 µm net) mounted in a Swinnex filter holder (Millipore, Burlington, MA, USA) as described in Palmini et al.⁸² . For immunofluorescence analysis, neurospheres were collected, concentrated in little volumes, picked up in drops placed on a slide and fixed.