Solvable

This experiment was conducted in 6- to 12-week-old male NTCP-KO mice and their respective C57BL/6JOlaHsd controls of the same age and sex. Animals were fasted 4–5 hours, after which they received an i.p. injection with Poloxamer 407 (1 mg/kg) to inhibit triglyceride hydrolysis by lipoprotein lipase. Mice were orally gavaged with olive oil containing tritium-labeled triolein (1 μCi/animal). The next 4 hours, for every 30 minutes 5 μL whole blood was sampled via the tail vein and collected in microscintillation vials containing 100 μL 0.1 M EDTA, 50 μL 3% H₂O₂, and 50 μL Solvable (Perkin Elmer). The experiment was terminated by heart puncture; organs were collected, weighted, and overnight dissolved in 1 mL Solvable (Perkin Elmer) at 37°C. Organ samples were decolored the next day by addition of 200 μL 30% H₂O₂, incubated for 1 hour at 50°C, and transferred to microscintillation vials. Radioactivity in organs and blood was measured by liquid scintillation counting using the TRI-CARB 2900 TR (Perkin Elmer). Plasma volume was estimated at 4.706% of total body weight (35 (link)–40 (link)).

Isotopically labeled (³⁵S) rHIgM12 was produced using Trans35S-Label (MP Biomedicals #51006) and validated for binding to the surface of primary cerebellar granule cell neurons in culture (Warrington et al., 2004 (link)). IgM was concentrated from culture supernatant by Polyethylene Gylcol and water precipitations, followed by re-suspension in normal saline and fractionation on a Superose-6 size exclusion column. Protein was quantitated by absorbance in a NanoDrop spectrophotometer and purity determined using denaturing and non-denaturing SDS PAGE. The acrylamide gel was exposed to autoradiography film to confirm radiolabel of light and heavy IgM chains. 1×10⁷ cpm (50 μg) of labeled IgM was administered intraperitoneally to groups of three 90-day-old SOD1G86R and age-matched normal FVB mice. Twenty hours later, plasma was harvested through the heart, mice were flushed transcardially with 50 ml of warm phosphate buffer and tissue acutely harvested. Tissues were weighed, a 50-100 mg portion dissolved in 0.5 ml of Solvable (PerkinElmer), incubated at 60°C for 4 h, 5 ml of Ultima Gold LSC liquid scintillation cocktail added per vial, mixed and cpm determined following the manufacturer's protocol. The ability of Solvable to lyse a given mass of tissue varies, so the manufacturer's guidance was used (LSC in Practice at Perkinelmer.com).

The TRA in plasma, whole blood, urine, and feces was determined by liquid scintillation counting. Plasma (0.2 mL) and urine (1 mL) samples were mixed directly with 10 mL liquid scintillation cocktail (Ultima Gold^TM, Perkin Elmer Inc., Waltham, MA, USA). To the whole blood samples (0.2 mL), 1 mL Solvable (Perkin Elmer Inc.), 0.1 mL 0.1 M EDTA, and 0.5 mL 30 % hydrogen peroxide were added to dissolve and decolorize the samples. Feces homogenates (0.2 mL) were first dissolved and decolorized using 1 mL Solvable (Perkin Elmer Inc.), 1 mL isopropanol, and 0.4 mL 30 % hydrogen peroxide. The decolorization reaction was started by warming the samples in a shaking water bath of approximately 43 °C, after which the samples were placed in a dark cool place for at least 1 h before liquid scintillation cocktail (10 mL) was added. Samples were counted on a Tri-Carb® 2800TR liquid scintillation counter (Perkin Elmer Inc.). Quench correction was applied with a calibration curve of quenched radioactive reference standards. Samples were counted to a sigma 2 counting error of 5 % or for 60 min at most. The lower limit of quantitation (LLOQ) was approximated to be 1.2 to 1.4 ng-eqv/g using the counting error as described previously [18 (link), 19 (link)].