*2.9. Immunofluorescence*

The slides were deparaffinized and rehydrated using conventional histological techniques. They were then rinsed with a PBS (0.1 M; pH 7.4). Nonspecific binding sites were blocked by incubating in IgG-free 2% bovine serum albumin (Sigma) for 30 min at room temperature (RT). Afterward, specimens were permeabilized with 0.2% Triton X-100 (Sigma, St. Louis, MO, USA) for 10 min at RT. Sections were then incubated overnight at 4 ◦C to 8 ◦C with primary antibodies HSP-60, -70, -90 (1:200 dilution), and caspase-3 (1:100 dilution) (all primary antibodies were obtained from

Santa Cruz Biotechnology Inc., CA, USA), followed by anti-rabbit Fluorescein Isothiocyanate (FITC) conjugated secondary antibodies and counterstained (1:100, Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) with VectaShield-DAPI (Vector Labs., CA, USA) for nuclei staining. The photomicrographs were taken near the site of the injection using a fluorescence microscope (Leica Microsystems, Wetzlar, GmBH, Germany) and the number of immunoreactive cells to HSPs and caspase-3 were quantified in the CA1 subfield of the Hp. All counting procedures were made blindly by an expert in morphology.

#### *2.10. Hematoxylin and Eosin (H&E)*

We evaluated the e ffect of EC on the neuronal loss caused by Aβ25–35 injection in the Hp by mean hematoxylin and eosin staining (H&E). The neurons were observed at 40× (Leica DM-LS, Leica Microsystems, Wetzlar, GmBH, Germany). Undamaged neurons were recognized as cells with round, blue nuclei and a clear perinuclear cytoplasm [19–24]. Damaged neurons were cells with changed nuclei (pyknosis, karyorrhexis, and karyolysis) and cytoplasmatic eosinophilia or loss of hematoxylin a ffinity.
