Stemi dv4 stereo microscope

After the final ABR test [see Auditory Function Evaluation (Auditory Brainstem Responses, ABR)], animals were deeply anesthetized (ketamine hydrochloride 25 mg/kg, xylazine 5 mg/kg, and acepromazine maleate 1.5 mg/kg body weight) and perfused transcardially with 0.9% NaCl (room temperature), followed by 4% paraformaldehyde at day 7 after surgery. The cochleae were quickly removed under a dissecting microscope (Zeiss Stemi DV4 Stereo Microscope, Jena, Germany) and processed for fluorescence and immunofluorescence analyses (described below). After 24 h of incubation with 4% paraformaldehyde in PBS at 4°C a pH 7.5, the cochleae were decalcified for 15 days (10% EDTA, changed daily), incubated for 48 h in cold 30% sucrose, and embedded in OCT. Frozen tissue samples were then cut into 16 μm thick sections using a Leica CM1850 cryostat (Leica Biosystems, Wetzlar, Germany). Five/ten neighboring randomly selected midmodiolus slices per cochlea were used to analyze the expression of transgenic GFP expression, as a reporter for ASC presence and survival; DAPI labeling was used to identify condensed cell nuclei. The analysis was performed using a fluorescence microscope equipped with a digital camera (Olympus BX63, Tokyo, Japan) and a confocal laser scanning system (TCS-SP2; Leica Microsystem, GmbH, Germany).

Adult female ticks (≥4.5 mm) [40 ] were counted on the left side of each animal, and the resulting value was multiplied by two to determine the parasite burden. This data were used to calculate the prevalence of tick infestation (TIP) (i.e., % of infested animals) and the intensity of tick infestation (TIR) (i.e., adult females/infested animals) in each group [41 ]. Tick counting was done between 7:00 to 8:00 a.m. [42 ]. The nymph/larvae ratio was compared between buffalo and bovine calves to determine the larval survival rate. For this purpose, the tick life stages were counted as described by Veríssimo et al. [43 ]; briefly, a 10 cm diameter area of the perineal region of each animal was scraped with a razor blade (Figure S1). The scraping material was collected and placed in tubes with a screw cap (15 mL) containing 3 mL of distilled water. The material was transferred to Petri dishes, and up to 100 ticks were counted on each plate using a Stemi DV4 Stereo microscope (Carl Zeiss, Oberkochen, Germany). In the counts, the numbers of larvae or nymphs were discriminated, and adult ticks were not considered. The nymph/larvae ratio was calculated as: Nymphs (%) = (nymphs/nymphs + larvae) × 100.

Images of small grains were determined using a Stemi DV4 Stereomicroscope (Brand Carl Zeiss Microscopy GmbH, Germany) with a zoom of 8X – 32X, while larger grains were taken with Scaner Laser Jet Pro (Model: 400 MFP, Series M425 DN, China), using a black background cover (0.25 m × 0.30 m), natural lighting and a 300x zoom. Shape and area analysis were performed from the images; the brightness, contrast and threshold of the images were adjusted and converted to 8-bit binary images to be analyzed using the particle analysis tool of ImageJ Software. ® (https://imagej.nih.gov/ij/). The area of the grains was calculated as the total area of the image [1 ,22 (link)]. The characteristics of the grains were evaluated according to what was described by Ubiergo & Lapp [23 ], contributions by Ferreira et al. [24 ], and Pablo-Pérez et al. [25 ], in addition, the terms to describe the shapes have been taken as reference from the descriptions used by Hoseney [26 ], Scade [27 ] and Kent [28 ]. The Remove BG online software was used to cut out the shapes, available at the following link https://pixlr.com/es/remove-background/.