Histology examinations

After MRI scanning and behavior test at week 13th, the rats were deeply anesthetized with a mixture of 15 mg/kg Zoletil and 9 mg/kg Rompun in saline. Then the rats were transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde. The brains were extracted and fixed overnight in the same postfixed solution, followed by dehydration in 30% sucrose.

For immunostaining, the rat's brains were dehydrated in a series of ethanol and embedded in paraffin. Coronal sections of the striatum were cut by a cryostat 10 µm thin and mounted on slides. After drying at room temperature overnight, the sections were deparaffinized and hydrated to distilled water. The sections then were incubated with serum blocking solution for 1 h and with mouse anti-HTT protein (1:200, clone mEM48, Merck, Darmstadt, Germany), mouse anti-ionized calcium-binding adapter molecule 1 (Iba-1) (1:200, Abcam, Cambridge, MA, USA), mouse anti-glial fibrillary acidic protein (GFAP) (1:200, Abcam), rabbit anti-oligodendrocyte transcription factor (Olig2) (1:200, Abcam), goat anti-NeuN (1:200, Abcam), and rabbit anti-cleaved caspase 3 (1:200, Abcam) antibodies overnight. The corresponding secondary antibody was applied before the sections were stained with DAB (Vector Laboratory, California, USA). Nuclei were counterstained with hematoxylin. Semi-quantification (a method for investigating protein expression and localization within stained tissues [29]) of different antibody-positive cells was performed by the ImageJ software (National Institutes of Health, Bethesda, MD, USA) with the following formula:

“antibody-positive cells (%) = (number of cells stained with antibody/total number of cells per field) × 100.

To observe the presence of iron, the sections were immersed in the mixed solution of hydrochloric acid and potassium ferrocyanide for 20 min and counterstained with nuclear fast red for 5 min. Finally, the sections were dehydrated and mounted with coverslips. To investigate the differentiation of injected stem cells, we stained the sections with anti-β-Tubulin III antibody (1:200, T8578, Merck), anti-GABA antibody (1:200, A2052, Merck), and anti DARPP32 antibody (1:200, ab40801, Abcam). Frozen sections were incubated with these primary antibodies at 4 °C overnight followings by incubating with Alexa Fluor® 488 (1: 500, ab150077 and ab150113, Abcam). The sections were counterstained with DAPI.