Thionin

Brain sections were mounted on slides and dried overnight under vacuum. Slides were washed (2×10 min) in KPBS, and then fixed for 20 min in 4% (w/v) PFA. They were then dehydrated according to the following sequence: H₂O, 50% (v/v) EtOH, 70% (v/v) EtOH, twice in 95% (v/v) EtOH, and thrice in 100% (v/v) EtOH; each dip carried out for 3 min), transferred next into xylene for 5, 30 and 2 min, and finally rehydrated using the reverse-order sequence (thrice in 100% (v/v) EtOH, twice in 95% (v/v) EtOH, and then 70% (v/v) EtOH, 50% (v/v) EtOH and H₂O; 2 min for each dip). Slides were next dipped 20 times in 0.25% thionin (Sigma-Aldrich). Mounted brain slides were dehydrated again (20 dips in each of H₂O, 50% (v/v) EtOH, and 70% (v/v) EtOH, and then 2 and 3 cycles of 3-min dips in 95% (v/v) and 100% (v/v) EtOH, respectively, and finally, 2 cycles of 3-min dips in xylene) before overlaying with coverslips in DPX mounting medium (Electron Microscopy Sciences).
For stereological analysis, 3 sections (−1.46, −2.06, and −2.46 mm from the bregma) were analyzed using the Stereo Investigator software. For each section, the cornu ammonis 1 through 3 (CA1/CA2, CA3) areas and the dentate gyrus area were defined and expressed as ratio of total hippocampus area for both hemispheres. Photographs were obtained with a Nikon C80i microscope equipped with a QImaging® color camera.

For anatomical overview, one series per brain was Nissl stained. After cryo-sectioning, the sections were mounted directly on SuperFrost Plus glass adhesion slides (Epredia), dried for 30 min, and kept at −20°C until staining. Sections were stained with 0.125% Thionin (Sigma-Aldrich, cat. no. 861340, USA) for 60 s, dehydrated in a graded series of ethanol solutions (1 min in 70%; 2 min in 96%; 2 × 5 min in 99%), and cleared for 5 + 10 min in xylene. Coverslips (24 × 50 mm, #0, Menzel-Gläser, Hounisen) were mounted with Eukitt^® Quick-hardening mounting medium (CAS. 25608-33-7, Sigma-Aldrich, Steinheim, DE).