Immediately after the last PET scan, brains were rapidly extracted and the hippocampus, cerebellum, and cortex were dissected. Tissue samples were then snap-frozen in liquid nitrogen and stored at −80 °C until used for western blot analysis.
Total protein was extracted from powderized brain tissue and 50 µg of protein were loaded and subjected to SDS-polyacrylamide gel electrophoresis according to a previously described procedure34 (link). Membranes were incubated overnight with the primary antibodies (SERT, target: sc-1458, dilution 1:500; secondary antibody: anti-goat IgG-HRP, sc-2020, dilution 1:2000; both Santa Cruz Biotechnology, Inc, Dallas, TX, USA; β-actin, housekeeping protein: A0760-40, dilution 1:2000; US Biological, Swampscott, MA, USA; secondary antibody: anti-mouse IgG-HRP, #7076, dilution 1:5000; Cell Signaling Technology, Danvers, MA, USA) used as the housekeeping protein. Chemiluminescent imaging was performed using a FluorChem HD2 imager (Alpha Innotec, Kasendorf, Germany) and densitometry values were determined using the software ImageJ (NIH, Bethesda, MD, USA). SERT protein densitometry values for each sample were normalized to the corresponding housekeeping protein value to obtain semiquantitative measures of SERT expression.
Total protein was extracted from powderized brain tissue and 50 µg of protein were loaded and subjected to SDS-polyacrylamide gel electrophoresis according to a previously described procedure34 (link). Membranes were incubated overnight with the primary antibodies (SERT, target: sc-1458, dilution 1:500; secondary antibody: anti-goat IgG-HRP, sc-2020, dilution 1:2000; both Santa Cruz Biotechnology, Inc, Dallas, TX, USA; β-actin, housekeeping protein: A0760-40, dilution 1:2000; US Biological, Swampscott, MA, USA; secondary antibody: anti-mouse IgG-HRP, #7076, dilution 1:5000; Cell Signaling Technology, Danvers, MA, USA) used as the housekeeping protein. Chemiluminescent imaging was performed using a FluorChem HD2 imager (Alpha Innotec, Kasendorf, Germany) and densitometry values were determined using the software ImageJ (NIH, Bethesda, MD, USA). SERT protein densitometry values for each sample were normalized to the corresponding housekeeping protein value to obtain semiquantitative measures of SERT expression.
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