# Hippocampal Neurons' Alignment on Quartz Grooves and Parylene Cues on Quartz Substrate
## 2 Materials and Methods
### Neurons Isolation, Seeding, and Cultivation  
Firstly, the test substrates were coated using poly-D-lysine (Sigma Aldrich, Shanghai, China). The 50 \(\upmu\)g\(\cdot\)ml\({}^{-1}\) poly-D-lysine was added on the test substrates, incubated for one hour, and aspirated. The test substrates were then rinsed using deionized water and prepared for neuron seeding. Dissection, dissociation, cultivation, and immunostaining of neurons was conducted according to an already optimized protocol. Hippocampal neurons (referred to hereafter only as neurons) were isolated from brains dissected from Sprague Dawley rat pups at the 18 th day of pregnancy. The brain tissue was minced in 3 mL of dissociation medium. The dissociation medium components at final concentrations were as follows: 97.5% of Hanks' Balanced Salt Solution Ca\({}^{++}\) and Mg\({}^{++}\) free (Sigma Aldrich, Shanghai, China), 0.11 mg\(\cdot\)mL\({}^{-1}\) of sodium pyruvate (Sigma Aldrich, Shanghai, China), 0.1% glucose, and 10 mM HEPES (Sigma Aldrich, Shanghai, China). The solution was transferred to a 15 mL tube and 1.5 mL of v and 0.5 mL of 0.25% Trypsin (Sigma Aldrich, Shanghai, China) were added. The tube was gently inverted five times, after which the tissue was left to settle to the bottom of the tube; this was repeated three times. Then, the brain tissue was incubated for an additional 5 min and gently inverted. Supernatant was removed and the tissue pellet was washed three times in 5 mL of dissociation medium each time the tissue pellet was left to settle to the bottom of the tube. After final wash, 5 mL of dissociation medium was replaced by 2 mL of fresh dissociation medium. The tissue was triturated seven times via fire-polished glass pipette. Large tissue pieces were left to settle to the bottom of the tube, and supernatant was transferred to a 50 mL conical tube. Then, 2 mL of dissociation medium was added into the tube with the large tissue pellet and trituration was repeated, while the supernatant was added to the 50 mL conical tube with previous supernatant. A total of 4 mL dissociated neurons should be obtained. The concentration of the dissociated neurons was determined, after which the neurons were diluted in a plating medium and seeded at prepared test substrates at a concentration of 2000 neurons per cm\({}^{2}\).  
The plating medium components at final concentrations were as follows: 86.55% of Minimum Essential Medium Eagle's (Sigma Aldrich, Shanghai, China) with Earle's Balanced Salt Solution (Sigma Aldrich, Shanghai, China), 10% of Fetal Bovine Serum (FBS; Sigma Aldrich, Shanghai, China), 0.45%, 1 mM of sodium pyruvate, 2 mM of glutamine (Sigma Aldrich, Shanghai, China), and 100\(\times\) Penicillin/streptomycin (Sigma Aldrich, Shanghai, China). After 24 h, half of the plating medium was replaced by a maintenance medium, the components of which at final concentrations were 96% Neuroblast medium (Thermofisher, Shanghai, China), 1x B-27 (Thermofisher, Shanghai, China), 2 mM glutamine, and 100x Penicillin/streptomycin. Half of the maintenance medium was replaced every four days. The neurons were cultivated in an incubator at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\).