Apotome 2 fluorescent microscope

The Nile Red staining method described by Rostron et al.⁹¹ was used with some modification. The yeast strains (2 × 10⁵ cells/ml in YPD medium), treated or untreated with the Xylaria extract or ECQ, were grown for 24 h at 30 °C with shaking. After that, 250 μL of cells were transferred into sterile 1.5 mL tubes to be stained. 25 μL of freshly prepared DMSO:PBS (1:1) and 5 μg/mL Nile Red in acetone were added, and the cells were incubated in the dark for 5 min at room temperature. The cells were washed twice with PBS and resuspended in 100 μL of 10% (v/v) formalin and fixed for 15 min in the dark at room temperature. The cells were washed twice with PBS and proceeded to imaging using a ZEISS Apotome.2 fluorescent microscope (ZEISS, Germany).

Animals were anesthetized and perfused transcardially with 5% sucrose followed by 4% paraformaldehyde (PFA). The brains were rapidly dissected and post-fixed overnight in 4% PFA at 4 °C. The brains were cryopreserved in 30% sucrose and then cut into 30-μm thick coronal sections. Hematoxylin and eosin (HE) staining was performed according to standard protocols to assess the volume of damage post-CCI. The stained sections were observed and acquired under light microscopy. For immunofluorescence staining, sections were first blocked with 5% bovine serum albumin (BSA) in 0.1% Triton X-100 for 1 h. The sections were then incubated over night at 4 °C with primary antibodies, rabbit anti-GFAP (1:250, Dako), rabbit anti-IBA1 (1:250, Wako), and rabbit anti-CD45 (1:150, Abcam) to mark astrocytes and microglia, respectively. Then, sections were incubated for 2 h with Alexa-594 (1:250, Invitrogen) secondary antibodies at room temperature. Immunofluorescence images were acquired using a Zeiss Apotome 2 fluorescent microscope. Fluorescent images were analyzed with Image J for quantification of fluorescent intensity and cell counting.

Cultured cells were rinsed with PBS and fixed in 2% paraformaldehyde (PFA) for 10 min at room temperature. Cells were subsequently permeabilized with 0.2% Triton X–100 for 10 minutes and incubated overnight at 4 °C with anti-MF20 (1/100, DSHB). Nuclei were stained with Hoechst (20 µg/mL, Sigma). To stain live cells, the latter were first washed with PBS and incubated 30 minutes on ice in anti-GFP antibody (Abcam, ab5450) diluted 1/800 in 3% BSA/PBS, followed by 10 minutes fixation with 4% PFA/PBS, 10 minutes permeabilization in 0.2% Triton/PBS and incubation with secondary antibody. Nuclei were then stained with Hoechst. All these cultures were visualized on a Zeiss ApoTome2 fluorescent microscope.