Eosin y 1 aqueous solution

20,000 cells were plated onto coverslips in 4-well plates and grown overnight. After washing once with PBS, cells were fixed with 4% PFA in PBS for 30 min at 4 °C. Slips were washed three times with PBS, once with ultra-pure water and stained with 500 µL Mayer’s hematoxylin (Bio-Optica, Milano, Italy) for 5 min at room temperature. To develop the staining, all wells were washed three times with 500 µL tap water followed by once with ultra-pure water. Eosin Y 1% aqueous solution (Bio-Optica, Milano, Italy) was added with 500 µL per well and stained for 3.5 min at room temperature. After washing twice with ultra-pure water, stained cells were dehydrated by dipping the coverslips three times into 90% EtOH and 12 times into xylenes (Sigma-Aldrich, St. Louis, USA) and mounted onto microscopy slides (Thermo Fisher Scientific, Waltham, USA) using Entellan Neu mounting medium (Merck, Darmstadt, Germany).

X-gal/FeCN-stained brains, embedded in paraffin, were sectioned at 16 µm and stained with Eosin Y 1% aqueous solution (Bio-Optica, 05-M10002). Images were obtained with the stereomicroscope MZ12 (Leica) equipped with color camera Leica DC500.