Dm2500

The mice were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and sequentially perfused with saline and 4% (w/v) PFA. The brains were subsequently removed and postfixed in 4% PFA at 4°C overnight. After cryoprotection of the brains with 30% (w/v) sucrose, coronal sections (40 μm) were cut on a cryostat (Leica CM1860, Germany) and used for immunofluorescence. The sections were incubated in 0.3% (v/v) Triton X-100 for 0.5 hour, blocked with 10% donkey serum for 1 hour at room temperature, and incubated with primary antibodies, including anti-c-Fos (1:500, rabbit, Santa Cruz Biotechnology, Dallas, TX), anti-glutamate (1:500, rabbit, Sigma-Aldrich), and anti-GABA (1:500, rabbit, Sigma Aldrich) at 4°C for 24 hours, followed by the corresponding fluorophore conjugated secondary antibodies for 2 hours at room temperature. Fluorescence signals were visualized using Leica DM2500 and Zeiss LSM710 microscopes and analyzed using ImageJ 1.4 (NIH). For counting immunoreactive cells, the 8-bit grayscale image was background subtracted before applying a threshold to all images. The threshold was adjusted within 10% of the average intensity, and cells at or above the threshold are considered immunopositive.

After the experiment, the mice were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and sequentially perfused with saline and 4% (w/v) paraformaldehyde (PFA). The brains were subsequently removed and postfixed in 4% PFA at 4 °C overnight. After cryoprotection of the brains with 30% (w/v) sucrose, coronal sections (40 μm) were cut on a cryostat (Leica CM1860) and used for immunofluorescence. Fluorescence signals expressed by the virus were visualized using a Leica DM2500 camera and Zeiss LSM710 microscope.

The cultures from the SC and SSC of P. chrysosporium were observed by fluorescence microscopy (Leica DM2500, Germany) and LSCM (Zeiss LSM 800, Germany). Before observation, the cultures were dyed using Nile red (0.5 g/L) and Calcofluor fluorescent stains (mixture of 1.0 g/L Calcofluor white and 0.5 g/L Evans blue) to highlight the cell wall. Subsequently, the intracellular lipid content of the fungal cells was estimated.

The frozen brain slides used for Nissl staining and hematoxylin and eosin (HE) staining were prepared in the same way as those used to immunofluorescence staining. Nissl staining was conducted to demonstrate the number of normal neuron in cortex and hippocampus as previously reported [28 ]. The frozen brain slides were immersed with 0.5% cresyl violet (Sigma-Aldrich, St. Louis, MO, USA) solution and dehydrated with 100% alcohol. Hematoxylin and eosin (HE) staining was performed using a HE Staining Kit (C0105; Beyotime Institute of Biotechnology, Haimen, China) to determine the morphological change. After sealed with neutral balsam, the slides were observed under a light microscope (DM2500; Carl Zeiss) by a blinded investigator.