Asp300

The collected organs including, aorta, liver, kidneys, spleen, adrenals, and brain samples were sliced into small sections of about 0.5–2 cm thickness and were fixed in 10% formalin for at least 48 hours. The fixed samples were placed in plastic cassettes and dehydrated using an automated tissue processor (ASP300; Leica Microsystems, Wetzlar, Germany). The tissues were embedded in paraffin wax (EG1160; Leica Micro-systems), and the blocks were trimmed and sectioned to 4 µm using a semiautomated microtome (RM2155; Leica Micro-systems). Then, the tissue sections were mounted on glass slides using a hot plate (HI1220; Leica Microsystems).
Subsequently, the tissue sections were deparafinized by two changes of xylene for 5 minutes each and rehydrated by three changes of different graded ethanol dilution (100%, 90%, and 70%) for 5 minutes each, respectively. Afterward, the sections were stained with Harris’s hematoxylin and eosin (HE) method, as described previously.28 Finally, tissue sections were mounted with glass cover slips using mounting medium distyrene-plasticizer xylene and were examined using a light microscope image analyzer (BX51TF; Olympus Corporation, Tokyo, Japan).

Cannula placements were examined after behavioral testing to ensure correct placement. Mice were anesthetized by intraperitoneal injection of chloral hydrate (450 mg/kg in a volume of 0.1 mL/10 g) and transcardially perfused with 2% paraformaldehyde (19 200, Electron Microscopy Sciences) in phosphate-buffered solution 1 × (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄) pH 7.4. Dissected brains were further fixed in Bouin’s fixative (Sigma-Aldrich) for 18–30 hours and then cleared and conserved in 70% (vol/vol) ethanol. Four transverse sections of each brain were obtained with an adult mouse brain matrix and processed together. Samples were processed using an automated tissue processor (ASP300, Leica Microsystem) and paraffin embedded. Briefly, samples were rinsed in bidistilled water for 30 minutes, then were gradually dehydrated through an ethanol (02860, Sigma-Aldrich) series (50%, 70%, 90%, 100%) and xylene (95 672, Sigma-Aldrich) at room temperature (RT) and finally embedded in paraffin (327 212, Sigma-Aldrich) at 62°C. Blocks were cut in 8–15 mm sections with a microtome (RM 2255, Leica Biosystems, Nussloch GmbH).