Chemikine bdnf and nt 3 sandwich elisa kit

After lethal pentobarbital injection, rats were craneotomized and brain samples were rapidly excised. The tissue samples were weighed and snap frozen in liquid nitrogen prior to storage at -70°C. Within two weeks of freezing, tissue samples were homogenized in ice cold homogenization buffer consisting of 100 mM Tris/Hcl, pH 7, 2% bovine serum albumin (BSA), 1 M NaCl, 4mM EDTA, 2% Triton X-100, 0.1% NaN₃, and the following protease inhibitors: 5 μg/mL aprotinin, 0.5 μg/mL antipain, 157 μg/mL benzamidine, 0.1 μg/mL pepstatin A and 17 μg/mL phenylmethyl-sulphonyl fluoride. Homogenates were prepared in approximately 20 volumes of the homogenization buffer to tissue-wet weight. The homogenates were centrifuged at 14,000xg for 30 minutes. The resulting supernatants were divided into two equal samples and used for the BDNF and NT-3 assay. The samples were analyzed by triplicate and following the instructions of the ChemiKine BDNF and NT-3 Sandwich ELISA Kit (Millipore, USA). Absorbance was measured in a microplate spectrophotometer at a 450nm wavelength (MultiSkan, Thermo Scientific, Finland).

After lethal pentobarbital injection, SC samples (2.5 cm including the site of injury) were rapidly excised. The tissue samples were weighed and snap frozen in liquid nitrogen prior to storage at −70 °C. Within 2 weeks of freezing, tissue samples were homogenized in ice cold homogenization buffer consisting of 100 mM Tris/Hcl, pH 7, 2 % bovine serum albumin (BSA), 1 M NaCl, 4 mM EDTA, 2 % Triton X-100, 0.1 % NaN₃, and the following protease inhibitors: 5 µg/mL aprotinin, 0.5 µg/mL antipain, 157 µg/mL benzamidine, 0.1 µg/mL pepstatin A and 17 µg/mL phenylmethyl-sulphonyl fluoride. Homogenates were prepared in approximately 20 volumes of the homogenization buffer to tissue-wet weight. The homogenates were centrifuged at 14,000×g for 30 min. The resulting supernatants were divided into two equal samples and used for the BDNF and NT-3 assays. The samples were analyzed by triplicate and following the instructions of the ChemiKine™ BDNF and NT-3 Sandwich ELISA Kit (Millipore, USA). Absorbance was measured in a microplate spectrophotometer at a 450 nm wavelength (MultiSkan, Thermo Scientific, Finland).