Fluoro-Jade B Staining of Neuronal Degeneration

The Fluoro-Jade B reagent was purchased from EMD Millipore (catalog no. AG310-30MG), and the staining protocol was followed as instructed by the manufacturer. Briefly, the staining solution was prepared from a 0.01% stock solution for Fluoro-Jade B that was made by adding 10 mg of the dye powder to 100 ml of distilled water. To make up 100 ml of staining solution, 4 ml of the stock solution was added to 96 ml of 0.1% acetic acid vehicle. This results in a final dye concentration of 0.0004%. The stock solution, when stored in the refrigerator, was stable for months, whereas the staining solution was typically prepared within 10 min of use and was not reused. Before staining, the asteroids were rinsed in distilled water and were then treated with 0.06% KMnO₄ solution for 15 min. Then, the asteroids were stained with Fluoro-Jade B working solution for 30 min followed by being washed with PBS twice for 5 min each. Brain assembloids were mounted in ProLong Gold antifade reagent (Thermo Fisher Scientific, catalog no. P36930) and stored in the dark until imaging.