Bx53 digital photomicroscope

Rostro-caudal sections of the dorsal horn of hippocampus (approximately from bregma − 1.06 to − 2.54 [58 ]) of each animal were mounted onto glass slide and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 1 min. Stained sections were viewed at low magnification by using Olympus BX53 digital photomicroscope. Digital images were then captured electronically and displayed on a computer screen. The volume of the total hippocampus and its subregions were unbiasedly estimated by means of point counting methods, using the Cavalieri’s principle [96 , 97 ]. Briefly, a 200-μm² point counting grid was superimposed on the images. The points hitting on the total hippocampus and its subfields (DG, CA1+CA3) were separately counted. The total volume of the hippocampus and its subfields was determined by applying the following formula:
$$\text{Volume}=\sum \mathrm{P}\mathrm{x}\mathrm{d}\mathrm{x}\mathrm{t}\mathrm{x}\mathrm{a}/\mathrm{p}$$ where “∑P” represented the number of points hitting the region analyzed, “d” was equal to the distance from one section to the next (d = 0.24 mm), “t” was the mean section thickness (t = 0.04 mm), and “a/p” was equal to the area associated with one point in the grid.

Volumes of the DG, Ammon horn CA1 and CA3 and whole hippocampus were estimated by quantitative light microscopy using the Cavalieri's method (Pakkenberg and Gundersen, 1997 (link)). In brief, rostro-caudal sections from hippocampus of each animal (taking every sixth serial section) were mounted onto glass slide and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 1 min. Stained sections were viewed at low magnification using Olympus BX53 digital photomicroscope. Digital images were then captured electronically and displayed on a computer screen. For each animal, DG, Ammon horn and whole hippocampus volumes were subsequently derived by multiplying the calculated mean surface area by the section thickness (40 μm) and the total actual number of sections in which the hippocampus was present.