The brain-like tissues were fixed with 4% paraformaldehyde for an hour and washed with 1× phosphate-buffered saline (PBS) 3 times at 5-min intervals. After being incubated in 1× PBS with 5% goat serum (AR0009, Boster, USA), a primary antibody diluted 1:200 and 0.3% Triton X-100 (T8200, Solarbio) overnight at 4 °C, the tissues were washed with 1× PBS 6 times at 10-min intervals and then incubated in 1× PBS with 5% goat serum, a secondary antibody diluted 1:200 and 0.3% Triton X-100 for 4 h before DAPI (AR1176, Boster) was added for an additional 10 min. Finally, the tissues were washed 6 times in 1× PBS at 10-min intervals and observed under an LSCM. All antibodies used are summarised in Table 2 . The LSCM software NIS-elements AR was used for measuring and counting the length and the number of axons and dendrites.
Summary of antibodies used in this study
| Cell type | Primary antibody | Secondary antibody |
|---|---|---|
| Neurons | Rabbit anti-MAP2 (8707S, CST, USA) | Goat anti-rabbit IgG, Cy3-conjugated (CW0159S, CWBIO, China) |
| Anti-neurofilament (2838S, CST) | Goat anti-mouse IgG, Cy3-conjugated (CW0145S, CWBIO) | |
| Astrocytes | Mouse anti-GFAP (3670S, CST) | Goat anti-mouse IgG, FITC-conjugated (CW0113S, CWBIO) |
| Rabbit anti-GFAP (16825-1-AP, Proteintech, USA) | Goat anti-rabbit IgG, FITC-conjugated (CW0114S, CWBIO) |