Cm3050s 3

Rat pups were anesthetized (3% isoflurane) at 48 h post-HI and transcardially perfused with ice-cold PBS and 10% formalin. The brains were collected and postfixed with 10% formalin at 4°C for 24 h. Thereafter, the brains were dehydrated in a 20% sucrose solution at 4°C until they sank and then transferred into 30% sucrose solution at 4°C until they sank. The brain samples were frozen at -80°C after being embedded into OCT compound (Scigen Scientific, USA); then, 10 μm thick coronal sections were cut at -20°C using a cryostat (CM3050S-3, Leica Microsystems, USA) and then mounted on glass slides for immunofluorescence staining, Fluoro-Jade C (FJC) staining, and MitoSox staining.

Nissl staining was conducted according to a previous report [6 ]. Briefly, the rats were treated as described above for immunofluorescence staining. The frozen brains were cut into 20 μm-thick coronal sections at − 20 °C with a cryostat (CM3050S-3, Leica Microsystems, USA). The sections were dehydrated in 95% and 70% ethanol for 1 min each, rinsed in tap water, and then distilled water for 30 s. Next, the sections were stained with 0.5% Cresyl Violet (Sigma-Aldrich, USA) for 3 min, and then washed in distilled water for 10 s and 30 s. The sections were then dehydrated in 100% ethanol and xylene two times for 1.5 min each, and covered with DPX (Sigma-Aldrich, USA). The sections were imaged using a microscope (Olympus-BX51) equipped with MagnaFire SP 2.1B software (Olympus). Each of the three sections per brain was averaged and then measured with ImageJ. The percentage of brain tissue loss = (contralateral hemisphere − ipsilateral hemisphere)/contralateral hemisphere × 100% [46 (link)].