Ds fi1c

Reversal rate measures the frequency at which a motile cell changes its direction (Toutain et al., 2005 (link); Caiazza et al., 2007 (link); Petrova et al., 2014 (link)). Bacteria expressing green fluorescent protein (GFP) were cultured overnight, sub-cultured and grown to the exponential phase (OD₆₀₀ of 1.0) before observation. The GFP-expressing bacteria were generated by introduction of a multicopy plasmid (pUCP-zsGreen1, GFP⁺) that constitutively expresses ZsGreen1 GFP under the control of the lac promoter (Li et al., 2017 (link)). Cultures were then diluted 1:100 into M63 medium (Kuchma et al., 2007 (link)) supplemented with 0.2% (w/v) glucose and containing 3% (w/v, low-viscosity/swimming conditions) or 15% (w/v) Ficoll (high-viscosity/swarming conditions) (Toutain et al., 2005 (link); Caiazza et al., 2007 (link)). Cells were monitored via fluorescence microscope (Eclipse Ni-U, Nikon) equipped with a 100×/1.45 oil objective lens and a Nikon DS-Fi1C camera. Real-time videos were captured using the NIS-Elements F Ver4.00.00 and Camtasia Studio V7.5 software package. The videos were subsequently analyzed to monitor individual cells for the number of times they reversed motility direction while within the field of view. Approximately 50 cells were measured for each strain and reversal rates are expressed as no. of reversals per cell per minute.

Hind paw fragments collected were fixed in Bouin’s solution, decalcified for 45 days in 5% EDTA, processed for paraffin embedding, sectioned (4 μm) and stained with hematoxylin-eosin (H&E) or sirius red by the picrosirius technique to assess the presence of collagen. Cellular profile was scored by counting the different cell types (macrophages, vacuolated macrophages, fibroblasts and lymphocytes) in H&E-stained paw sections, analyzed with a photomicroscope (Olympus, Miami, FL, USA) at a final magnification of 200x. Five images from each mouse were captured using Motic Images Plus v.2.0 (Motic China Group Co. Ltd., Xiamen, China). These images were divided into four quadrants, two of which were randomly selected for cell quantification with ImageJ 1.45 s software (NIH, USA—2011). Collagen quantification of the lesion site was determined in sirius red-stained paw sections under polarized light using a photomicroscope (Nikon Eclipse 80i) with a camera (Nikon DSFi1C) coupled to a computer using Nis Element software (Shinjuku, Japan), at a final magnification of 200x. Four images of four sections from each mouse were considered for the study and analyzed by Image Pro Plus (version 4.5). The results were expressed as percentage of area with the presence of collagen compared to the total measured area.

To test if an isolate could be a pheromone producing recipient or a potential donor, cells were grown for 16 h in 20 ml of TSB. Cells were pelleted by centrifugation (10,000 RCF) for 15 min at 4 °C. Supernatant was removed and filter sterilised (Millipore 0.22 micron filters) providing a pheromone-enriched broth. The clumping assay consisted of 500 μl pheromone-enriched broth, 500 μl fresh TSB, and 20 μl of a 16 h culture added in a 1.5 ml Eppendorf and incubated for four hours (37 °C rotating at 150 rpm). From the final suspension, 20 μl was dropped on a glass slide and a coverslip (22 mm) was applied. Clumping was determined by eye. Isolates that induced clumping but did not clump in the presence of other supernatants were deemed potential recipients. Isolates that readily clumped were characterised as potential donors. Cells were imaged using phase contrast with 100x (Nikon plan fluor 1.3 oil ph3 DLL) on a Nikon eclipse E400 with a Nikon DS-fi1c. Images were captured with NIS-elements and imageJ (NIH).

Histological sections of liver were stained with: (a) hematoxylin and eosin (H&E) and (b) periodic acid-Schiff (PAS) for histopathological evaluation; (c) sirius red by picrosirius technique to assess the presence of fibrosis; (d) impregnated by silver-methenamine in accordance with Gomori-Grocott and counter-stained with light green or H&E [25] (link), [26] (link) for locating the yeast in the hepatic parenchyma; and (e) immunostained with anti-gp43 antibody to detect expression of the glycoprotein gp43.
Expression of gp43 in the liver was detected by avidin-biotin peroxidase immunohistochemical staining method using polyclonal primary antibody, anti-gp43 obtained in rabbits, and diluted in a concentration of 1∶50, and revealed using a immunohistochemical commercial kit (Histostain-Plus kits, Invitrogen 2^nd Generation, LAB-AS Detection System, Camarilo, CA).
Slides were observed and photographed using a trinocular light microscope (Nikon Eclipse 80i), with a camera (Nikon DSFi1C) coupled to a computer using Nis Element software (Shinjuku, Japan).

SOD expression in the digestive diverticula were detected immunocytochemically based on the methods described by Takata et al. [17 (link)], with modifications. First, the digestive diverticular were fixed in 4% paraformaldehyde, dehydrated in ethanol, and then embedded in paraffin. In brief, paraffin-embedded sections were deparaffinized in xylene, rehydrated in ethanol, and then incubated overnight at 4 °C with primary mouse anti-SOD antibodies (dilution 1:2000; TA326384, OriGene, Rockville, MD, USA), as well as with secondary antibodies afterwards (HRP-conjugated anti-mouse immunoglobulin; 1:1000). The antibody binding was visualized by applying 3,3′-diaminobenzidine as a detection system. Slides were counterstained and mounted with Canada balsam for observation under a light microscope (DM 100; Leica, Wetzlar, Germany); images were captured with a digital camera (DS-Fi1c, Nikon, Tokyo, Japan).