Dpx mounting reagent

The coronal sections of the brain containing the dorsal hippocampus were prepared at 10 μm thick via a cryostat (Thermo Scientific™ HM525 Cryostat). In brief, the brains were removed and fixed with 4% paraformaldehyde (Sigma-Aldrich, USA) in 0.1 M phosphate buffer pH 7.4, 4°C overnight. Then, they were infiltrated with 30% sucrose (Merck, Germany) solution for 48–72 hours. Following the mentioned process, the serial sections of the brains were prepared at 10 μm thick and placed on slides coated with 0.3% aqueous solution of gelatin containing 0.05% aluminum potassium sulfate (Sigma-Aldrich, USA). Then, they were stained with 0.25% cresyl violet (Sigma-Aldrich, USA), dehydrated through graded alcohols (70, 95, 100%; 2x) (RCI Labscan, Thailand), placed in xylene (Merck, Germany), and mounted using a DPX mounting reagent (Merck, Germany). The assessment of neuron density in the prefrontal cortex and CA1, CA2, CA3 and the dentate gyrus of the hippocampus was performed under an Olympus light microscope model BH-2 (Japan) at 40x magnification. Counts were performed in three adjacent fields, and the mean number was calculated. The results were expressed as the density of neurons per 255 μm² [23 (link)].

Matrigel invasion assays were performed in a 24-well plate using 2 × 10⁵ cells re-suspended in 200 μl of serum-free media. These cells were added to the upper chambers and 600 μl of conditioned media (1:1 ratio of conditioned and fresh serum containing media) were added in the lower chamber. The inner side of the insert with 0.8 mm membrane (BD Falcon, USA) was pre-coated with 15 μl of Matrigel (Corning, USA). After 24 hours, the inserts with cells on the outer side of the membrane were fixed with 4% para-formaldehyde, stained with 1% crystal violet (Sigma-Aldrich, USA), and mounted on slides using D.P.X mounting reagent (Merck, USA). Images were taken using an Olympus SZ61 stereo microscope with a 10X objective.