47 adpms e01

Fasted serum insulin (10-1247-01, Mercodia, Uppsala, Sweden) and total and HMW adiponectin (47-ADPMS-E01, Alpco, Salem, NH, USA) were measured in dams at GD 18.5 using ELISA. Male or female fetal serum was pooled from each dam and total adiponectin was measured with mouse DuoSets ELISA (DY1119, R&D Systems, Minneapolis, MN, USA). Serum TG levels were analyzed using a TR210 kit (Randox, Crumlin, UK), with the following changes to the manufacturer’s instructions: 200 µL of reagent R1 was added to each well containing 2 µL standard, or serum, in duplicates. A plate reader (SpectraMax I3x, Molecular Devices, San Jose, CA, USA) was used to measure the absorbance.

A small tail blood sample was taken to quantify serum insulin levels by ELISA (Mouse insulin 10–1247-01, Mercodia, Sweden) and glucose was measured with a blood glucose meter (Contour XT, Bayer Health Care, Mishawaka, USA). A larger blood sample for serum analyses was collected right after decapitation. Total and high molecular weight adiponectin in serum was analyzed by ELISA (47-ADPMS-E01, Alpco, Salem, NH, USA) with a sensitivity of 0.032 ng/mL and an intra- and inter-assay CV < 4%. In another cohort of mice, cerebrospinal fluid (CSF) was collected by puncturing the cisterna magna using a glass capillary with a fine tip and gentle suction^{41 (link)}. Samples with suspected blood contamination were immediately discarded. Total adiponectin was analyzed in serum (diluted 1:50,000) and CSF (diluted 1:100) using an adiponectin ELISA (80569, CrystalChem, Zaandam, The Netherlands) with a sensitivity of 0.008 ng/mL and an intra-assay coefficient variation (CV) < 10%. The absorbance was measured on a plate reader (SpectraMax I3x, Molecular Devices, San Jose, CA, USA).