Eukitt

For histochemical staining (COX/SDH), muscle cross sections on glass slides were incubated in COX medium (100 μM cytochrome c, 4 mM diaminobenzidine tetrahydrochloride, and 20 μg/mL catalase in 0.2 M phosphate buffer, pH 7.0) for 90 min at 37 °C. Sections were then washed in standard PBS, pH 7.4 (2 × 5 min), and incubated in SDH medium (130 mM sodium succinate, 200 μM phenazine methosulphate, 1 mM sodium azide, 1.5 mM nitro blue tetrazolium in 0.2 M phosphate buffer, pH 7.0) for 120 min at 37 °C. Finally, they were washed in PBS, pH 7.4 (2 × 5 min), rinsed in distilled water, and dehydrated in an increasing ethanol series up to 100%, prior to incubation in xylene and mounting in Eukitt (Merck, Burlington, MA, USA). For skeletal muscle fiber-type identification, antibodies against different myosin heavy chain isoforms were used. Anti-myosin heavy chain ‘slow’ antibody was used to identify the MyH7 isoform in type I fibers (Merck, Burlington, MA, USA)). For type IIa fibers, we used myosin A4.74 antibody (obtained from the Developmental Studies Hybridoma Bank, developed by Helen M. Blau, The University of Iowa). For sequential staining we used a diaminobenzidine staining kit as well as an alkaline phosphatase staining kit (both from Abcam, Cambridge, UK).

MG63 cells were seeded on UV-sterilized coverslips laid at the bottom of 6-well plates (1.5 × 10⁵ cells per well); after 24 h (50% ≤ confluence ≤ 70%), cells were treated with the GNp, GN1p, GN2p and CS powders. After treatments, the medium was removed and coverslips were washed twice with D-PBS, afterward cells on coverslips were fixed by overnight incubation at 4 °C with 4% (w/v) paraformaldehyde (PFA) in D-PBS. The day after, a standard hematoxylin/eosin (HE) staining protocol was performed as follows: (1) D-PBS washing (3×, 10 min each); (2) washing (1×) with distilled water for 5 min; (3) hematoxylin staining for 15 min and quick washing with distilled water (1×); washing with running water for 15 min; (4) rapid washing with distilled water and staining by eosin (acidified with glacial acetic acid) for 1 min; (5) after washing with distilled water, dehydration by increasing concentrations of ethanol in alcohol/distilled water solutions (50%, 70%, 95% and 100% v/v, 2 min each); (6) rapid immersion in xylene (2 min) and (7) mounting on microscope slides with the Eukitt^® (Merck KGaA, Darmstadt, Germany) acrylic resin mix and 12–18 h drying before final image acquisition. Bright/fluorescence images of samples were acquired with a Nikon AZ-100 stereoscopy system equipped with the Nikon (Miniato, Japan) NIS-Elements D package software.

Mice were perfused with 4% paraformaldehyde dissolved in 0.1 phosphate buffer, pH 7.3 24 hours after the first KA injection, post-fixed overnight in the same fixative, and cryoprotected in 30% sucrose. 30 μm-thick coronal brain sections were obtained in a freezing microtome (Leica, Wetzlar, Germany). Sections containing dorsal hippocampus (Bregma = −1.2 to −1.990 ) were rinsed for 2 h in 0.1 M Tris, pH 7.4, mounted and air dried at room temperature overnight. The next day, sections were pre-treated for 3 min in absolute ethanol, followed by 1 min in 70% ethanol and 1 min in distilled water. They were then oxidized in a solution of 0.06% KMnO4 for 15 min. After three rinses of 1 min each in distilled water, the sections were incubated for 30 min in a solution of 0.001% Fluoro-Jade B (Chemicon) containing 0.01% of DAPI (Sigma) in 0.1% acetic acid. The slides were rinsed in deionized water for 3 min each, dried overnight, cleared in xylene, cover-slipped with Eukitt (Merck, Darmstadt, Germany) and examined using an Olympus (Hamburg, Germany) BX61 epifluorescence microscope. The statistical analysis of the obtained data was performed using Bonferroni post hoc test (Multiple comparison test) using Prism 5.0c (Mac OsX, Grahpad). Data are presented as mean ± standard error of the mean (S.E.M.). A value of ***P < 0.01 was considered statistically significant.

Acetic ethanol- or PFA-fixed paraffin sections were deparaffinized, rehydrated using a xylene/ethanol gradient, stained with haematoxylin (#3870, JT Baker^®, Phillipsburg, NJ) and eosin-Y alcoholic (#102439, Merck, Darmstadt, Germany), Sirius Red (Direct Red 80, #365548, Sigma), or using the Herovici procedure [16 (link)], and mounted with Eukitt^® (#03989, Sigma).

Apoptosis detection was conducted via TUNEL assay kit, following the instructions. Briefly, the paraffin-embedded tissues used for TUNEL assay were sliced into 4-μm-thick sections and then dewaxed in xylene. After infiltration in 3% hydrogen peroxide for 15 min, sections were placed in TUNEL reaction mixture for 0.5 h and treated with converter-POD solution for 1 h. Sequentially, 3,3-diaminobenzidine tetra-hydrochloride (DAB) and hematoxylin dye were applied to stain and visualize the slides. TUNEL staining was performed on five consecutive sections for one specimen. After processing and dehydration, the slides were mounted with Eukitt (Sigma-Aldrich) and observed under a light microscope (OLYMPUS 1 × 70-SIF2, Japan). Cells with brownish-yellow particles in nuclei were considered as apoptotic cells. The total number of TUNEL-positive staining cells in the hippocampal CA1 region was calculated in three randomly selected views from each section and five sections per sample under a light microscope with 400 × magnification. Apoptosis index was acquired via the following formula: apoptosis index (AI) = the number of apoptotic cells/the number of total cells × 100%.