2.4. TUNEL Assay

For visualization of neuronal apoptosis in the hippocampus, we performed terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to assess apoptotic cell death in rats treated by vehicle, paclitaxel, thalidomide, and paclitaxel and thalidomide. In brief, the rats were anesthetized and perfused with 200 ml of 0.9% normal saline with 0.1% of heparin, followed by 200 ml PBS containing 4% paraformaldehyde. Brains were removed and placed in 4% paraformaldehyde to postfix for 24 h at room temperature. Then, the brains were dehydrated and sectioned into brain slices at a thickness of 16 μm. An In Situ Cell Death Detection Kit (TMR Green; Roche Applied Sciences, Indianapolis, IN) was used to determine the number of apoptotic cells. Ten sections randomly selected from each group were fixed in ethanol-acetic acid at −20°C. The sections were incubated with proteinase K, rinsed, and incubated with 3% H₂O₂ at room temperature. Then, 0.5% Triton X-100 was used to permeabilize the cell membrane followed by incubation with the TUNEL reaction mixture (6 μl per section) at 37°C. The sections were then visualized using Converter-POD with 0.02% 3,3′-diaminobenzidine (DAB; 100 μl/section) at room temperature. Then, the sections were counterstained with hematoxylin for 30 seconds and washed again with running water for 10 minutes. Cell counting was performed in the CA1 subfield of the dorsal hippocampus. Cells with brown nuclei were considered apoptotic and were analyzed by using the Image-Pro 6.0 software (Media Cybernetics Inc., Rockville, MD, USA). The number of TUNEL-positive cells was quantified under ×400 magnification, and the density of stained cells was presented as cells per square millimeter.