Thunder imager tissue microscope

A randomly chosen series was used to calculate the total volume of the DG in each animal using Nissl staining. Slices were mounted on 2% gelatine-coated glass slides and air-dried at rt for 48 h. The slides were sequentially immersed in the following solutions: 6 min in toluidine blue, 10 sec in bi-distilled water, 2 min in EtOH 70°, 2 min in EtOH 96°, 2 min in EtOH 100°, 2 min in EtOH 100°, and 2 min in Xylene (PanReac, #251769). Next, sections were embedded in DePex (Serva Electrophoresis™, #1824301) and dried for 48 h at rt before imaging. Images were acquired under a THUNDER Imager Tissue microscope equipped with a Leica DFC9000 GTC VSC-09991 camera (Leica Microsystems Ltd., Wetzlar, Germany) and using a 5X dry objective. Images were processed using the Leica Application Suite X (LAS X) software provided by the manufacturer (Leica Microsystems Ltd., Wetzlar, Germany). The DG volume was determined using the freehand selection tool in Fiji software to measure the granule cell layer (GCL) plus the SGZ area on each section of the series. Each area was multiplied by the thickness of the tissue (namely 50 µm) and by the sampling fraction (8). The numbers obtained were summed to calculate the total DG volume, which is expressed in mm³.

For the morphological evaluation, 12 μm RST sections were stained with hematoxylin and eosin (H&E) according to the standard protocol.
To quantify the melanin content, 12 μm thick RST sections were subjected to Fontana–Masson staining. The sections were then treated with a 2.5% aqueous silver nitrate solution for 10 min, 0.2% aqueous gold chloride for 1 min, and 5% aqueous sodium thiosulfate for 5 min. The melanin levels were normalized by counterstaining the epidermis with fast red.
To detect the expression of photoaging-related proteins, 12 μm frozen sections were hydrated in distilled water, subjected to antigen retrieval by heating in a citrate buffer (pH 6), and blocked with 3% bovine serum albumin (Sigma-Aldrich) prepared in PBS. The sections were incubated overnight in a humidified chamber at 4 °C with the following primary antibodies: anti-TRP-2 (1:100), anti-filaggrin (1:100), anti-collagen type I (1:500), and anti-laminin-5 (1:200). The sections were then incubated for 2 h with secondary antibodies, namely 1:1000 Alexa Fluor 488 goat anti-rabbit IgG (Abcam) or Alexa Fluor 555 goat anti-mouse IgG (Abcam); diluted in PBS; and mounted using a mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). Images were obtained using a Leica THUNDER Imager Tissue microscope and analyzed using the ImageJ Software.