Syn per reagent protocol

Synaptosomal enriched preparations were extracted from 50 mg of human DLPFC or 60 mg of dissected rat cortex using Syn-PER reagent protocol (Thermo Scientific, Rockford, IL) following the manufacturer instructions. We pooled brain tissue from the 2 rats used for each condition to obtain a single preparation per time point and temperature. Three fractions (S1, P1, P2) from each preparation were isolated, stored at −80 °C, and used for downstream protocols as specified below. The S1 fraction contains soluble cytosolic elements. The P2 fraction is enriched in synaptosomes. The P1 fraction, which contains mostly nuclei, myelin, and large non-homogenized tissue, was used as a reference for synaptosomes enrichment in P2 fractions by Western Blot analysis. All the procedures of centrifugation were performed at 4 °C using proteinase inhibitors (Thermo fisher, cat #A32955) to reduce proteolysis and denaturation. P1 and P2 fractions were re-suspended in Syn-Per buffer solution and the amount of protein was quantified by DeNovix QFX fluorometer instrument (DeNovix Inc. Wilmington, USA) and Qubit reagents (Invitrogen, cat# Q33212).

Synaptosome enriched preparations were isolated from 50 mg of brain cortex from four rats using the Syn-PER reagent protocol (Thermofisher, Waltham, MA, USA). Briefly, 50 mg of cortex from each rat was homogenized in Syn-PER reagent to which was added an EDTA free protease inhibitor (Thermofisher, Cat#. A32955). Synaptosomes were enriched in the P2 fraction after the last centrifugation, with P1 and S1 fractions enriched in myelin, nuclei, and any non-homogenate tissue or cytosolic elements. Resulting P2 proteins were re-suspended in Syn-PER and quantified via fluorometry using a DeNovix QFX unit (DeNovix, New Castle, DE, USA) and flowcytometry using a Guava EasyCyte (Guava Soft v2.7, Luminex, Austin, TX, USA) [35 (link)]. For the preparation of cerebellar synaptosome enriched preparations, 50 mg of the whole cerebellum was used following the same procedure described above.