Tissue samples (liver, heart, neck) obtained during dissection of embryos on 9ED of CHEST were frozen, stored (−80 °C) and then analyzed in order to establish the acetylcholinesterase activity induced by applied venoms obtained from the B. arietans and B. parviocula snake species.
Samples were homogenized (Sonoplus mini20, Bandelin, Berlin, Germany) in cold buffer according to manufacturer instructions. The composition of buffer was as follows: 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate, Phosphatase inhibitor cocktail, Protease inhibitor cocktail and PMSF (all Sigma-Aldrich, St. Louis, MO, USA). The tissue homogenates were analyzed using colorimetric Acetylcholinesterase Assay Kit (AChE; Abcam, Shanghai, China). The activity of AChE was recorded according to the manufacturer’s instructions and adapted for reading on microplates for an ELISA device (λ 410 nm; Multiskan®EX Spectrometer, Thermo-Fisher, Abingdon, UK). AChE hydrolyzed acetylthiocholine to thiocholine and acetate, and then the amount of thiocholine was quantified by DTNB (5,5′-Dithiobis(2-nitrobenzoic acid)) reagent, which was proportional to the AChE activity. The data obtained were normalized to protein values (mU/mg protein).
Samples were homogenized (Sonoplus mini20, Bandelin, Berlin, Germany) in cold buffer according to manufacturer instructions. The composition of buffer was as follows: 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate, Phosphatase inhibitor cocktail, Protease inhibitor cocktail and PMSF (all Sigma-Aldrich, St. Louis, MO, USA). The tissue homogenates were analyzed using colorimetric Acetylcholinesterase Assay Kit (AChE; Abcam, Shanghai, China). The activity of AChE was recorded according to the manufacturer’s instructions and adapted for reading on microplates for an ELISA device (λ 410 nm; Multiskan®EX Spectrometer, Thermo-Fisher, Abingdon, UK). AChE hydrolyzed acetylthiocholine to thiocholine and acetate, and then the amount of thiocholine was quantified by DTNB (5,5′-Dithiobis(2-nitrobenzoic acid)) reagent, which was proportional to the AChE activity. The data obtained were normalized to protein values (mU/mg protein).