Rats were perfused with PBS. Brains were immersed in Golgi staining solution which contained 50% potassium dichromate (MP 021563389), 5%
Mercuric chloride (Sigma‐Aldrich, M1136), and 5%
Potassium chromate (Sigma‐Aldrich, 529508) and protected from light. Brain sections with thickness of 150 μm were made at 7 days after incubation. When performing Golgi staining, sections were washed with distilled water, dehydrated with ethanol, and then treated with ammonia (3 : 1). The sections were subsequently washed by distilled water and incubated in 5% sodium thiosulfate for 10 min, and then dehydrated with degraded ethanol and clarified with xylene. Then, the sections were observed under bright field of Olympus
FV1000 (Tokyo, Japan). The images were taken by z‐stack scanning with the excitation wavelength of 405 nm, and the virtual color was converted into green color. For spine density and subtype analysis, dendrites were reconstructed by using IMARIS and the spines were grouped according to the following criteria as described (Kang
et al. 2017). Tubby: length (spine) < 1.5 and max width (head) < mean_width (neck) *1.2; mushroom: max width (head) > mean width (neck) *1.2 and max_width (head) > 0.3; if the spine was not classified as mushroom or stubby, it was defined as long‐thin.
Liang L., Xie R., Lu R., Ma R., Wang X., Wang F., Liu B., Wu S., Wang Y, & Zhang H. (2020). Involvement of homodomain interacting protein kinase 2‐c‐Jun N‐terminal kinase/c‐Jun cascade in the long‐term synaptic toxicity and cognition impairment induced by neonatal Sevoflurane exposure. Journal of Neurochemistry, 154(4), 372-388.
