Horse serum

The sections were taken from the knee joint for apoptosis dyeing of chondrocytes. Briefly, the paraffin was removed and sections were rehydrated by soaking in xylene (100%), alcohol (100, 95, 75 and 50%), and then proteinase K (20 mM; Sigma-Aldrich; Merck KGaA) was added for 30 min and rinsed with PBS. Hydrogen peroxide (3%) solvent was added for 10 min. Sections were permeabilized with permeabilization solution containing 0.1% Triton X-100 and 0.1% sodium citrate for a 30 min reaction at room temperature and rinsed with PBS. Horse serum (3%, Jackson ImmunoResearch Inc.) was added for 60 min and the TUNEL reaction mixture agent (TUNEL reaction mixture; In Situ Cell Death Detection kit, Fluorescein; cat. no. 11684795910, Sigma-Aldrich; Merck KGaA) for a 90-min reaction at 37˚C. Samples were stained with DAB dye at 25˚C for 5 min, and then soaked in 10 mg/ml hematoxylin stai (Merck KGaA) at 25˚C for nuclear staining for 90 sec. Slides were analyzed under a light microscope with a microscope digital image output system. The percentage of TUNEL-positive cells were quantified by determining the number of positive chondrocytes with x200 magnification images, as previously described (42 (link)).

U87 and MG-63 cells were adjusted to a density of 1.0× 10⁴ cells/cm² and seeded on the nanotube coatings with or without annealing. After 24 hours, the cells were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde for 20 minutes. The cells were blocked with horse serum (1:20 dilution, Jackson ImmunoResearch, West Grove, PA, USA) for one hour at room temperature, immunolabeled with the primary antibodies anti-rabbit caspase-3 (1:100 dilution, Bioss Inc, Woburn, MA, USA) and anti-mouse acetylated α-tubulin (1:150 dilution, Abcam, Cambridge, MA, USA) overnight at 4°C, washed with phosphate-buffered saline three times, and immunolabeled with the appropriate secondary antibodies (fluorescein isothiocyanate conjugation for acetylated α-tubulin and tetramethylrhodamine-5-(and 6)-isothiocyanate conjugation for caspase-3, 1:50 dilution, Jackson ImmunoResearch) for one hour in the dark. The cells were washed in phosphate-buffered saline. The coverslips were mounted on slides with ProLong^® Gold Antifade Mountant and 4,6-diamidino-2-phenylindole (DAPI; Invitrogen, Life Technologies, Carlsbad, CA, USA) before storage at 4°C. The cells were imaged using a Leica confocal microscope (Leica, Wetzlar, Germany). Fluorescence signals were captured after excitation with 355, 488, and 543 nm laser lines.