Cm 3050 s

All tissues in O.C.T were cut at 10 µm on a cryostat (LEICA CM 3050 S) and put on slides (Thermo Scientific). These slides were conserved at −20 °C. The epitope revelation protocol was realized with PBS incubation at room temperature for 15 min. Then, we let the slide in methanol (Fisher Chemical) at −20 °C for 2 h and incubated in formaldehyde 2% at room temperature (Sigma) for 15 min. We saturated non-specific sites by BSA 4% (Sigma) during 2 h at room temperature and washed for 1 h with PBS. The primary antibodies (Supplementary Data 4; 1:200) were added overnight at 4 °C. Then we washed the slide during 1 hour in PBS and we added secondary antibodies (Supplementary Data 4; 1:200) and DAPI (Supplementary Data 4; 1:1000) for 1 h at 4 °C. We washed the slide during 1 h in PBS and we added mounting medium (Invitrogen). We finally dropped off the cover (Fisher Scientific, Dutscher) from the slide with mounting medium. The fluorescence staining was observed and captured by a spinning-disk on three tissue sections per monkey (Yokagawa, CellVoyager CV1000). We analyzed these images by ImageJ (Fiji).

The presence of flavonoids, such as quercetin aglycone and derivates, was corroborated by their natural epifluorescence observed in brain areas after histological analysis. Rats receiving quercetin or rutin at 30 and 100 mg/kg, i.p., were euthanized and their brains were quickly dissected on dry ice and stored at −80 °C until histological analysis. Rostro–caudal coronal sections from bregma −0.60 mm to −2.92 mm [26 ] were cut at a 20 µm of thickness on a cryostat (Leica Biosystems Nussloch GmbH CM3050S, Nussloch, Germany), adhered to Superfrost/Plus slides (Fisher Scientific, Waltham, MA, USA), each 120 µm apart and stored at −80 °C until used. Thawed sections were observed with epifluorescence using a Leica DM 1000 LED fluorescence microscope with a HI PLAN 10X/0.25 or 40X/0.65 objective and bandpass filter sets of 340–380/dichromatic mirror 400/barrier LP 425 for DAPI emission (Leica Microsystems AG, Wetzlar, Germany). The images were captured using a digital FireWire camera (DFC450C, Leica Biosystems Nussloch GmbH, Nussloch, Germany) and Leica Application Suite software. Sections were exposed to DAPI blue filter sets, and the resulting images were processed in Adobe Photoshop CS6 software (Adobe Inc., San Jose, CA, USA) using an iMac computer.

Hippocampi were derived from postmortem brains collected at autopsy at the Cuyahoga County Medical Examiner’s Office, Cleveland, OH. Tissues were dissected and rapidly frozen in 2-methylbutane on dry ice without fixation and kept in dry ice before permanent storage at –80°C. Postmortem hippocampus samples from 20 subjects diagnosed with SUD, 24 with comorbid SUD and MDD, 20 with MDD, and 20 psychiatrically-normal control donors were obtained from the University of Mississippi Medical Center Postmortem Brain Core. Hippocampi were cut into 14 μm thick sections using a cryostat (Leica CM3050S) and mounted on Fisher Superfrost slides.