3 3 diaminobenzidine (dab)

We detected apoptosis in the lung tissues in each group using a TUNEL staining kit (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, the paraffin sections were dewaxed to hydration and treated with 3% H₂O₂ and proteinase K liquid (20 mg/mL) in succession, followed by TUNEL reagent for 60 minutes at 37°C. The sections were then stained with 100 μL 3,3′-diaminobenzidine (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) for 10 minutes at 25°C and counterstained with hematoxylin. TUNEL-positive cells in each section were identified and counted under an Olympus IX 70 inverted fluorescence microscope (Olympus) at 200× magnification. The data were presented as the percentages of total endothelial cells that were TUNEL-positive.

Spinal cord segments L_4–6 obtained 1, 2, 4, and 8 weeks after curcumin administration were fixed for 3 days in 10% neutral formaldehyde before being used for immunohistochemical staining of S100. Briefly, L_4–6 spinal cord slices were placed in 3% H₂O₂ for 10 minutes to block endogenous peroxidase, and then boiled for 10 minutes in 0.01 M sodium citrate buffer (pH 6.0). Slices were then blocked for 15 minutes with anti-species serum and washed with 0.01 M phosphate-buffered saline with Tween 20 (pH 7.4). Rabbit anti-mouse S100 polyclonal antibody (1:500; Beyotime Institute of Biotechnology, Najing, Jiangsu Province, China) was added and incubated at 4°C overnight. Biotinylated goat anti-rabbit IgG (1:1,000; Boster, Wuhan, Hubei Province, China) was added for 20 minutes at 37°C. Streptavidin and biotinylated horseradish peroxidase were added for 20 minutes at 37°C. The labeled antigens were visualized using 3,3′-diaminobenzidine (Jiancheng Bioengineering Institute, Nanjing, Jiangsu Province, China). Brown staining indicated positive protein labeling. Microscopic examination (PM-10A0, Olympus, Beijing, China) was conducted by two pathologists who were blinded to treatments and used Image-Pro Plus 6.0 software (Media Cybernetics Inc.) to analyze the integrated optical density by scanning the stained area.