R540ip

Mice were anesthetized by 1% isoflurane (R510-22, RWD, China) using a gas anesthesia machine (R540IP, RWD, China) and subsequently perfused by injecting 4% paraformaldehyde (60 ml) through the heart. The brain was removed and fixed with 4% paraformaldehyde. The left damaged brain tissue was dissected to embed in paraffin and then sectioned using a microtome (RM2016, Leica, Germany) with a thickness of 5 μm. The sections were baked in an oven at 60 °C and dewaxed, and then stained with Harris hematoxylin for 3–8 min. After this, the sections were stained with eosin for 1–3 min after washing twice with tap water. Finally, the sections were dehydrated twice with 95% alcohol (5 min each) and washed three times with xylene (5 min each), and then fixed with natural gum. All sections were scanned with a digital pathology scanner system (Pannoramic 250, 3D HISTECH Ltd., Hungary), and 5 fields in injured areas were randomly selected for analysis. The output digital pathological images were 400x enlarged.

Pheasant chicks (190–210 g) and BALB/c mice (20–22 g) of either sex were anesthetized by isoflurane inhalation with an anesthesia respirator (R540IP, RWD Life Science). The head was fixed, and the scalp was cut longitudinally to expose the skull of the mouse. A gentle saline drip over the exposed surgical opening prevented dehydration of the skull. The blood flow in the cerebral cortex of the animals was monitored by a laser-speckle blood flow imaging system (Version 2.0, RFLSI Pro, RWD Life Science, Shenzhen, China, 2017) before and after the injection of the toxins.

Mucetin and stejnulxin were purified as previously reported [9 (link),25 (link),57 (link)]. BALB/c mice (6-week-old) of either sex were anesthetized under anesthesia respirator (R540IP, RWD Life Science) as the manufacturer’s instructions, and perfused with normal saline 30 min after tail vein injection of mucetin and stejnulxin. Liver, lung and kidney were fixed with 4% formalin, dehydrated by 40% sucrose solution and then cut into 10-µm-thick sections on a freezing microtome (CryoStar NX50 OP, Thermofisher, Waltham, MA, USA). The sections were subsequently stained with hematoxylin and eosin for further analysis as previously reported [62 (link)]. Animal protocol in this work, SMKX2017026, was reviewed and approved by the Animal Care and Use Committee at Kunming Institute of Zoology, Chinese Academy of Sciences in December 2017.