Heparin coated capillary tubes

Blood was collected utilizing the retro-orbital eye bleed procedure prior to the first dose (pre-dose) and then at various time points post dose. For this procedure, the mice were anesthetized using isoflurane. Heparin-coated capillary tubes (Fisher Scientific) were inserted into the posterior corner of the mouse eye; the tube was inserted at a 45° angle to ∼1 cm and rotated until the blood from the retro-orbital sinus was released. Approximately 200 μl was collected from the left eye of each mouse according to the IACUC protocol for blood collection. The blood was collected in Becton Dickinson (BD) serum separator tubes. Serum samples were kept at room temperature for 1 h and then spun in a microcentrifuge at 22 × g at room temperature for 10 min. Serum was transferred to 1.5-ml microcentrifuge tubes. Serum collected for the analysis of transthyretin was stored at –80°C until sample processing. Serum collected for the analysis of circulating C5 was kept at room temperature for 15 min and then immediately transferred to 4°C prior to spinning in a microcentrifuge at 22 × g at room temperature for 10 min.

Trunk blood of male and female offspring was collected at E18.5 or E19, and postnatally at 1 h, 3 h, or P1 following a vaginal delivery or at 3 h following a C-section delivery. Blood was collected in heparin-coated capillary tubes (Fisher Scientific), transferred into clean microcentrifuge tubes, and centrifuged at 1680×g at 4 °C for 5 min. Plasma was collected and stored at -80 °C until analysis. Plasma samples were diluted twofold in nanopure water and osmolality was measured using the Advanced Micro Osmometer (Advanced Instruments, Norwood, MA).