Isoflurane gas

The AEP inhibitor, also named δ-secretase inhibitor 11 (7-Morpholin-4-yl-benzo [1,2,5]oxadiazol-4-ylamine, PubChem CID: 1095027) [37 (link),45 (link)], was purchased from J&K Scientific Ltd. (Beijing, China). The δ-secretase inhibitor 11 was first dissolved in dimethyl sulfoxide (DMSO) to make a stock solution and then diluted in a 0.9% NaCl solution containing gum arabic for systemic treatment. The ELISA kits for detecting IL-6 (70-EK206/3-96) and IL-1β (70-EK201B/3-96) were obtained from MULTISCIENCES Lianke Biotech, Co. Ltd. (Hangzhou, China). The ELISA kit for TNF-α (H052-96T) was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The primary antibody for mature AEP was obtained from R&D Systems Inc. (Minneapolis, MN, USA). Antibody for microtubule-associated protein 2 (MAP2) was obtained from Abcam (Cambridge, MA, USA). Antibodies for SOX2 (sc-365823) and Nestin (sc-23927) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Isoflurane gas was purchased from RWD Life Science (Shenzhen, China).

Tumours were measured by bioluminescence using an IVIS Spectrum CT Imaging System (PerkinElmer, Waltham, MA, USA), which captured the luminescence signal emitted from the engrafted tumour. Prior to imaging, the mice were anaesthetized with inhalation of isoflurane gas (RWD Life Science) and injected i.p. with 150 mg·kg⁻¹d‐luciferin potassium (J&K Scientific) aqueous solution. The isoflurane was balanced with oxygen and dialled to 2.0% for the induction of anaesthesia and 1.0% for maintenance. Images were subsequently captured 10 min following injection. Signal intensity was quantified within a region of interested over the head, as defined by Living Image software. All images represent 2‐min exposure time, and the average number of photons·s⁻¹·cm⁻² per steradian were recorded. The data were analysed using Living Image 4.4.5 software (PerkinElmer, RRID:SCR_014247).