Dfc7000

The brains were harvested and immersed in 4% paraformaldehyde (Fisher) overnight. Then, brains were washed 3X with sterile saline for 1 h, embedded in paraffin, 4 μm coronal sections were serially cut using a cryostat (Tanner Scientific, model: TN50), fixed onto glass slides, and subjected to hematoxylin & eosin or Periodic acid-Schiff staining to examine tissue or fungal morphology, respectively. GXM (MAb 18B7 is an anti-cryptococcal GXM IgG1 generated and generously provided by Arturo Casadevall at the Johns Hopkins Bloomberg School of Public Health; 1:1,000 dilution) and Iba-1 (rabbit anti-human Iba-1; 1:1,000 dilution; FujiFilm Wako) specific Ab (conjugated to horseradish peroxidase; dilution: 1:1,000; Santa Cruz Biotechnology) immunostaining to assess capsular release and distribution and microglial phenotype, respectively, near cryptococcomas. The slides were visualized using a Leica DMi8 inverted microscope, and images were captured with a Leica DFC7000 digital camera using LAS X digital imaging software. GXM distribution in tissue sections at 10X magnification (n = 15 fields per brain) was calculated using NIH Image J color deconvolution tool software (version 1.53q). The mean color intensity of the GXM for each treatment group was plotted in Prism version 9.5 (GraphPad). The images were examined and analyzed by Dr. Mohamed F. Hamed, a veterinary pathologist.

The neuroprotective potential of apigenin was assessed with the Fluoro-Jade B assay (FJB, Millipore, AG310). This staining was used to evaluate neuronal death. Cells were cultured in 96-well black bottom plates (Corning Incorporated, 3603) and treated as described. After treatments the co-culture, supernatants were removed and the cells were fixed with ethanol at 4°C for 10 min, washed three times with PBS, and permeabilized with 0.3% Triton X-100 in PBS (Merck) for 10 min. After this time, the cultures were washed three times with distilled water and incubated with 0.001% FJB solution for 30 min at room temperature (RT), under slow agitation and protected from the light. After incubation, the cells were washed three times with PBS and incubated for 5 min at RT in the dark with 5 μg/ml 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining, and then washed three times with PBS. Analyses were performed on a spectrophotometer (Varioskan™ Flash Multimode Reader, Thermo Plate), and the fluorescence intensity of each sample was measured at 480 nm for FJB and 350 nm for DAPI. The value of absorbance of FJB of each well was normalized to the DAPI absorbance in the same well. Three independent experiments were performed. Thereafter, cells were analyzed using a fluorescence microscope (Leica, DFC7000). Quantification was analyzed with ImageJ 1.33u.

Kidneys were fixed in 4% paraformaldehyde, embedded in paraffin and sectioned. A terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) assay was performed using One Step TUNEL Apoptosis Assay Kit (Beyotime) following the manufacturer’s protocol. Mounted samples were analyzed by Automatic Fluorescence Microscope (Leica DFC7000).

Proliferation was evaluated using bromodeoxyuridine (BrdU) (Sigma Aldrich, Saint Luis, MO, USA). BrdU (10 µM) was added to the wells at the start of each treatment. Cells were fixed and DNA was denatured by treatment with denaturing solution (2 N HCl) for 20 min at room temperature. Mouse anti-BrdU monoclonal antibody (1:200, Sigma Aldrich, Saint Luis, MO, USA, B8434) diluted in PBS was pipetted into the wells and allowed to incubate for 1 h. Unbound antibody was washed away and cells were incubated with Alexa Fluor 594 antibody specific for mouse IgG (1:500, Invitrogen - Molecular Probes, Eugene, OR, USA) diluted in PBS-T for 1 h under slow agitation at room temperature. After incubation, the cell nuclei were stained with DAPI (5 µg/mL) for 10 min at room temperature. All reagents were used in accordance with the manufacturer’s instructions. Experiments were performed in triplicate. Thereafter, cells were analyzed using a fluorescence microscope (Leica, Wetzlar-Germany, DFC7000). Quantification was analyzed with ImageJ 1.33u (Bethesda, MD, USA).

To evaluated proliferation was using the Bromodeoxyuridine (BrdU) cell proliferation assay (Sigma–Aldrich, Inc., St. Louis, MO, USA). BrdU (10 μM) was added to the wells since each treatment had started. Cells were incubated for 2 h in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. Cells were fixed and DNA was denatured by treatment with denaturing solution (2 N HCl) for 20 min at room temperature. Anti-BrdU monoclonal antibody (mouse, 1:200, Sigma–Adrich, St. Louis, MO, USA B8434) diluted in PBS was pipetted into the wells and allowed to incubate for 1 h. Unbound antibody was washed away and cells were incubated with Alexa Fluor 546-conjugated goat anti-mouse IgG (1:500; Molecular Probes, A11003), diluted in PBS-T for 1 h under slow agitation at room temperature. After incubation, the cell nuclei were stained with DAPI (5 μg/ml) for 10 min at room temperature. All reagents were used following the manufacturer’s instructions. Experiments were performed in triplicate. Thereafter, cells were analyzed using a fluorescence microscope (Leica, DFC7000) Quantification was analyzed with ImageJ 1.33u software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Reduced glutathione (GSH) is an important antioxidant compound for CNS cells, as it serves as a substrate for peroxidases and is conjugated with free radicals [13 (link)]. Here, we evaluated the protective effect of the flavonoid agathisflavone on LPS-induced cytotoxicity in primary microglia culture, through the determination of intracellular GSH. For this, the cells were seeded at a density of 3 × 10⁴/cm², and after stimulation with LPS and treatment with agathisflavone, the cells were washed three times with PBS and incubated with 400 μL of medium containing 1 mM of monochlorobimane (MCB) (Sigma Aldrich, St. Louis, MO, USA) for 30 min in the dark. L-buthionine-S-R-sulfoximine (BSO) was used at a concentration of 1 mM as an inhibitor of GSH synthesis and adopted as a positive control. After the incubation time, the cells were washed again with PBS. The detection of free glutathione was performed using a fluorescence microscope (Leica, DFC7000, Wetzlar, Germany). Three independent experiments were carried out.