Ultra turrax t8

Powdered, frozen samples were weighed and homogenized using a hand-held polytron homogenizer (Ultra-Turrax T8; IKA Labortechnik, Staufen, Germany) in ice-cold RIPA buffer containing 25 mM Tris-HCl (pH 7.4), 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 1% sodium deoxycholate, 5 mM EDTA, 150 mM NaCl, protease inhibitor cocktail (Complete Mini; Roche, Basel, Switzerland), and phosphatase inhibitor cocktail (PhosSTOP; Roche). Homogenates were centrifuged at 10,000× g for 15 min at 4 °C, and supernatants were collected and used for Western blot analyses.

A cylindrical sample was taken from the central part of the lens in an anterior-posterior direction using a corkborer with an internal diameter of 3.5 mm. The lens samples were placed in 500 μl lysis buffer (150 mM NaCl, 50 mM Tris–HCl (pH = 7.4), protease inhibitor (Roche 04 693 124 001) and phosphatase inhibitor (Roche 04 906 837 001)). The lenses were mechanically homogenized with an Ultra-Turrax T8 (IKA Labortechnik) and left to lyse for 30 minutes on ice. Samples were lyophilized over night. After lyophilization the lens samples were kept at −80° C until they were analyzed for the concentration of advanced glycation end products.

Three-month-old ALZ17 mice were anaesthetized with a mixture of ketamine and xylazine and placed on a heating pad to maintain body temperature. Mice were stereotactically injected into the right hippocampus (A/P: −2.5 mm from bregma; L: −2.0 mm; D/V: −1.8 mm) using a Hamilton syringe. Brainstem homogenates of 6-month-old paralysed homozygous P301S mice and 3-week-old paralysed P301SxTAU62^on mice were prepared for inoculation in ALZ17 mice. Brainstems were weighed and diluted 1:10 in phosphate-buffered saline (PBS) for seeding. After dilution, samples were homogenized using an Ultraturrax T8 (IKA Labortechnik) and sonicated briefly (Bandelin SONOPULS; 90% power, 10% cycle, 10-s pulses). Homogenates were then centrifuged at 4000g for 20 min at 4°C, and aliquots of the supernatant were stored at −70°C for later usage. During inoculation, each mouse received 5 μl of brainstem homogenate at a speed of 1.25 μl/min. Following injection, the needle was kept in place for an additional 3 min before withdrawal. The surgical area was cleaned with sterile saline and the incision sutured. Mice were monitored until recovery from anaesthesia, post-interventional analgesia was administered, and animals were checked regularly following surgery. After 20 months of incubation, the seeded mice were sacrificed.