Nis elements microscope imaging software

Under urethane anesthesia, rats were perfused through the aorta with saline for 2 min, followed by 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4, for 10 min. The intact rats were stored overnight at 4°C. This was followed by brain extraction and 50‐μm‐thick horizontal sections were cut in 0.1 mol/L Tris buffer, pH 7.6, using a vibratome. Serial sections were mounted on Superfrost Plus slides for subsequent Nissl (1% cresyl violet) and Fluoro‐Jade B staining (Schmued and Hopkins 2000). Subsequently, specimens were dehydrated with ethanol, the ethanol replaced by xylene, and coverslipped with Permount. All histological and staining procedures have been described previously (Bumanglag and Sloviter 2008). Brightfield and fluorescence specimens were imaged digitally using a Nikon E800M microscope with a Nikon DS‐Fi2 digital camera and Nikon NIS‐Elements microscope imaging software. Images were prepared for display using GIMP 2.8.18 software (GNU Image Manipulation Program, open‐source from the GIMP team at www.gimp.org).

Cells were seeded into a confocal dish (coverglass-bottom dish). After pretreatment with vehicle or samples for 6 h, the cells were co-treated with 100 μM H₂O₂ for 1 h. The cells were further incubated with JC-1 (chloride salt; Biotium, Hayward, CA, USA) staining solution (5 μg/mL) at 37 °C for 15 min and rinsed with culture media. MMPs were estimated by measuring the fluorescence of free JC-1 monomers (green) to JC-1 aggregates in mitochondria (red) as observed through a Nikon ECLIPSE TE2000-U microscope (Nikon Instruments Inc., Tokyo, Japan), and the quantification of the red and green fluorescence intensity ratio in individual cells was performed using Nikon NIS-Elements microscope imaging software.

Collected faecal samples or large intestine contents collected at necropsy were stored at 4 °C for a maximum of 3 days until analysis. All samples (n = 146) were analysed by: (i) the combined sedimentation/flotation technique using a 44% zinc chloride solution (specific density [S.D.] = 1.3) as flotation medium [25 ] and (ii) the sodium acetate-acetic acid-formalin (SAF)—concentration technique [25 ]. In addition, when sufficient material was available (n = 121), the Baermann–Wetzel technique was performed [25 ]. Parasite stages were photographed and measured using a digital image processing system (Nikon NIS-Elements—Microscope Imaging Software; Nikon Corp., Tokyo, Japan) and identified through morphological characterization [25 ]. Isospora-type oocysts were further differentiated into Toxoplasma-like and Cystoisospora species by their respective size [25 ], and Toxoplasma-like oocysts were further differentiated by PCR. Eggs of Taenia spp., Hydatigera spp., and Echinococcus spp. were grouped as taeniid eggs and further differentiated by PCR.