Ivis spectral in vivo imaging system

Luciferase‐stable LN229 cells (5 × 10⁵) in a total volume of 10 μL of PBS were implanted into the frontal lobes of nude mice (female; 5 weeks old; Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China) using a stereotactic device (KDS310; KD Scientific, Holliston, MA, USA). The burr hole was positioned 1 mm anterior and 2 mm lateral from the anterior fontanel, and the injection depth was adjusted to 2.0 mm. For the panobinostat treatment groups, panobinostat was administered intraperitoneally at a dose of 10 mg/kg three times a week for 4 weeks. At days 7, 14 and 21 after implantation, tumor growth was examined using bioluminescence imaging (IVIS Spectral In Vivo Imaging System, PerkinElmer; Hopkinton, MA, USA). Body weight was measured every 3 days, and the mice were sacrificed when they began to show symptoms of continuous discomfort. The brains of mice were removed, fixed, embedded in paraffin and sectioned for IHC staining.

To eliminate possible bias, five nude mice (male, 4 weeks old; GemPharmatech Co., Ltd; Nanjing, China) were randomly assigned to each group. A human glioma cell line expressing luciferase (3 × 10⁵ cells suspended in 10 μL PBS) was implanted into the frontal lobes of nude mice using a stereotactic device (KDS310, KD Scientific; Holliston, MA, USA). At days 7, 14, 21 and 28 after implantation, tumor growth was examined using bioluminescence imaging (IVIS Spectral In Vivo Imaging System, PerkinElmer; Hopkinton, MA, USA). Animals were euthanized by the cervical dislocation method when they exhibited any signs of persistent discomfort, such as severe hunched posture, reduced activity, apathy, leg dragging, or weight loss of more than 20%. The brains of the mice were perfused, and subsequent analysis was performed on sections with hematoxylin and eosin staining, and IHC/IF.