Hypothalamic Tissue Fixation and Sectioning

The hypothalamic tissue blocks were first rinsed briefly with running tap water and then, immersion- fixed with 4% formaldehyde in 0.1 M phosphate buffer saline (PBS; pH 7.4) for 7–14 days. The fixed hypothalami were trimmed in a way to include the optic chiasm rostrally, the mammillary bodies caudally and the anterior commissure dorsally [19] (link), [20] (link), [25] (link), [27] (link), [28] (link). Sagittal cuts were made 2 cm lateral to the midsagittal plane on both sides. Subsequently, the blocks were cut in halves and infiltrated with 20% sucrose for 5 days at 4°C. The right hemihypothalami were placed in a freezing mold, surrounded with Jung tissue freezing medium (Leica Microsystems, Nussloch Gmbh, Germany; diluted 1∶1 with 0.9% sodium chloride solution), snap-frozen on powdered dry ice, and stored at −80°C until sectioned at 30 µm in the coronal plane with a Leica SM 2000R freezing microtome (Leica Microsystems). The sections were stored permanently in anti-freeze solution (30% ethylene glycol; 25% glycerol; 0.05 M phosphate buffer; pH 7.4) at −20°C.