Vt1000p vibratome

5–9 weeks old male Sprague-Dawley rats were used in this study. Rats were anesthetized by intraperitoneal injection of a mixture of ketamine and xylazine. Under deep anesthesia, determined by cessation of toe-pinch reflex, rats were transcardially perfused with ice-cold cutting solution containing (in mM) 210 sucrose, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 0.5 CaCl₂, 7 MgCl₂, 7 dextrose and 3 Na-pyruvate (all from Sigma Aldrich). After transcardial perfusion, rats were decapitated quickly and the brain was surgically removed in the presence of ice-cold cutting solution. 350-μm thick near-horizontal middle (Bregma −6.5 mm to −5.1 mm) hippocampal slices were prepared using VT1000P vibratome (Leica) in the presence of oxygenated ice-cold cutting solution. Slices were submerged in a holding chamber containing oxygenated chamber solution composed of (in mM): 125 NaCl, 2.5 KCl, 1.25 NaH₂PO₄, 25 NaHCO₃, 1 CaCl₂, 2 MgCl₂, 10 dextrose and 3 Na-pyruvate, and were incubated at 34 °C for 10–20 minutes and then at room temperature for at least 45 minutes before recording.

All animal work was performed according to the guidelines of the Institutional Animal Care and Use Committee at the Fourth Military Medical University. Carefully prepared slices containing the SNc (300 μm thick) were separated from male Sprague-Dawley rats (12–14 days of age) as described previously (Xue et al., 2012 (link)). Briefly, animals were sacrificed, and the brain was rapidly removed and immersed in ice-cold modified ACSF [in mM: 11 glucose, 126 NaCl, 18 NaHCO₃, 1.2 NaH₂PO₄, 2.5 KCl, 2.4 CaCl₂, 1.3 MgCl₂; bubbled with a gas mixture (95% O₂/5% CO₂; pH 7.4)] for more than 30 min. Then, the midbrain was blocked in a coronal plane, fixed to the stage of a VT1000P vibratome (LEICA, Germany) and sliced. We maintained slices at room temperature for 1 h before experimentation.

For mouse 4T1 breast tumor production, a million tumor cells suspended in DPBS were transplanted into the mammary fat pad of female BALB/c mice orthotopically, as described previously [29 (link)]. After 14 days, mice were sacrificed under deep anesthesia and tumors were collected. Live 4T1 tumor slices were then sectioned at the thickness of 200 μm using a Leica VT1000P vibratome. Tumor slices were cultured in the 12-well plate (VWR, West Chester, PA, USA, Cat# 82050-930) and incubated with AgNP-555 or AgNP-555 + TP peptide in the FBS-free DMEM medium at 37 °C for 2 h. After incubation, the slices were etched, washed by DPBS, fixed with 4% PFA and mounted with the DAPI-containing mounting medium by coverslips. Nikon C2 confocal (Melville, NY, USA) was used to examine the tumor slices.