14c inulin

[¹⁴C]Urate (55.0 mCi/mmol), [³H]guanine (10.7 Ci/mmol), [³H]hypoxanthine (27.0 Ci/mmol), [³H]thymine (65.0 Ci/mmol), and [³H]uracil (42.8 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA), [¹⁴C]inulin (1.9 mCi/g) was from American Radiolabeled Chemicals (St. Louis, MO), and [³H]polyethylene glycol 4000 (PEG 4000, 1.5 mCi/g) was from PerkinElmer Life Sciences (Boston, MA). Unlabeled urate, guanine, hypoxanthine, thymine, and uracil were obtained from Wako Pure Chemical Industries (Osaka, Japan), and Ko143 was from Sigma‐Aldrich (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Wako Pure Chemical Industries and Invitrogen (Carlsbad, CA), respectively. Mouse monoclonal antibodies for the tag peptides of DYKDDDDK (FLAG) and hemagglutinin (HA) were obtained from Wako Pure Chemical Industries (product numbers of 014‐21881 and 018‐22381, respectively, for the anti‐FLAG and anti‐HA antibodies), and a mouse monoclonal antibody for β‐actin (product number A5441) and horseradish peroxidase‐conjugated goat anti‐mouse IgG (product number A8924) were from Sigma‐Aldrich. All other reagents were of analytical grade and commercially obtained.

The in-vivo 125I-Aβ40 (PerkinElmer; Boston, MA, USA) clearance was investigated using the brain efflux index (BEI) method as we described previously [20 (link)]. In brief, at the end of the treatment, animals were intraperitoneally anesthetized with xylazine and ketamine (20 and 125 mg/kg, respectively), followed by the insertion of a stainless-steel guide cannula into the right caudate nucleus, an area rich with blood microvessels, of the mice’s brain following previously established protocols [58 (link)]. A tracer fluid (0.5 μL) containing 125I-Aβ40 (30 nM; PerkinElmer, MA, USA) and 14C-inulin (0.02 mCi, American Radiolabeled Chemicals, St. Louis, MO, USA), prepared in an artificial extracellular fluid buffer (ECF; 122 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.4 mM CaCl2, 1.2 mM MgSO4, 0.4 mM K2HPO4,10 mM D-glucose, and 10 mM HEPES, pH 7.4), was microinjected in the mice brains. Thirty minutes later, the brains were rapidly collected for a 125I-Aβ40 analysis. 125I-Aβ40 and 14C-inulin radioactivity was determined in the brain tissues using Wallac gamma and beta counters (PerkinElmer Inc. Waltham, MA, USA), respectively. 125I-Aβ40 BEI was determined using the formula: