Eclipse e200

Immunocytochemistry (ICC): The SH-SY5Y cells were seeded on the poly-l-lysine coated coverslips, and treatment was given for 24 h. ICC was performed in control, treated and transfected cells, as reported previously [30 (link)]. The signals were captured through fluorescent (Nikon eclipse E200, Japan) or confocal microscopy (Carl Zeiss, Jena, Germany).
Immunohistochemistry (IHC): After the treatment, rats were sacrificed, and the brain was perfused with 15–20 mL of PBS. Quickly, each brain was isolated and kept in 4% sucrose solution for 3 h, incubated in 10% sucrose solution for another 3 h, and finally, shifted to 30% sucrose solution and kept overnight at 4 °C. The brains were then stored at −20 °C for 2 h, blocks were prepared with cryomatrix (Invitrogen, Waltham, MA, USA), and sections were cut down using a cryostat (Thermo Scientific, Waltham, MA, USA) to the thickness of 10–15 microns. The sections were collected on the poly-l-lysine coated slides immediately and processed for staining. Finally, the sections were mounted using an anti-fade medium containing counter stain DAPI (nuclear stain), and images were captured by fluorescent microscope (Nikon eclipse E200, Japan).

After collection, the stool samples were subjected to coproparasitological analysis using two techniques for researching helminth eggs and protozoan oocysts: Willis fluctuation [31 (link)] and spontaneous sedimentation by Hoffman et al. [32 ], adapted according to Hoffmann [33 ].
For flotation, a hypersaturated NaCl solution (NaCl 35%) was added to the stool samples and subsequently filtered through a sieve and gauze to remove debris, subjecting it to spontaneous fluctuation due to the difference in density of eggs and oocysts. Finally, they were observed between the slide and coverslip (with the addition of a drop of Lugol’s solution) using an optical microscope (Nikon Eclipse E200, Nikon, Tokyo, Japan).
The sedimentation test was carried out with the addition of water to the sample, filtered through a sieve and gauze to remove debris, subjected to spontaneous sedimentation in a specific cup, and then observed between the slide and coverslip (with the addition of a drop of Lugol) using optical microscopy (Nikon Eclipse E200). Eggs and oocysts were identified according to the method of Zajac and Conboy [34 ].

Spermatozoa motility was assessed using a computer-assisted sperm assay (CASA) method according to World Health Organization guidelines (46 (link)). After euthanasia, spermatozoa were collected from the cauda epididymis of mice and suspended in Dulbecco’s modified Eagle medium (DMEM)-F-12 medium with 10% FBS and incubated at 37.5°C for 30 min; samples were then placed in a prewarmed counting chamber. The Microptic sperm class analyzer (CASA system) was used in this investigation. It was equipped with a 20-fold objective, a camera adaptor (Eclipse E200; Nikon, Japan), and a camera (acA780-75gc; Basler, Germany), and it was operated by an SCA sperm class analyzer (Microptic S.L.). The classification of sperm motility was as follows: grade A linear velocity, >22 μm s⁻¹; grade B linear velocity, <22 μm s⁻¹; curvilinear velocity, >5 μm s⁻¹; grade C curvilinear velocity, <5 μm s⁻¹; and grade D, immotile spermatozoa. The spermatozoa motility data represented only grade A and grade B since only these two grades are considered to be functional.

Spermatozoa motility was assessed using a computer-assisted sperm assay (CASA) method according to World Health Organization guidelines (46 (link)). After euthanasia, spermatozoa were collected from the cauda epididymis of mice and suspended in Dulbecco’s modified Eagle medium (DMEM)-F-12 medium with 10% FBS and incubated at 37.5°C for 30 min; samples were then placed in a prewarmed counting chamber. The Microptic sperm class analyzer (CASA system) was used in this investigation. It was equipped with a 20-fold objective, a camera adaptor (Eclipse E200; Nikon, Japan), and a camera (acA780-75gc; Basler, Germany), and it was operated by an SCA sperm class analyzer (Microptic S.L.). The classification of sperm motility was as follows: grade A linear velocity, >22 μm s⁻¹; grade B linear velocity, <22 μm s⁻¹; curvilinear velocity, >5 μm s⁻¹; grade C curvilinear velocity, <5 μm s⁻¹; and grade D, immotile spermatozoa. The spermatozoa motility data represented only grade A and grade B since only these two grades are considered to be functional.

One mL samples of culture were fixed with 1 μL Lugol’s iodine solution. The fixed samples were mixed to homogeneity, placed in a hemacytometer (QiuJing factory, Shanghai, China), and counted under a microscope (Eclipse E200, Nikon, Tokyo, Japan). The counting was performed six times for each sample and cell concentration of each sample was calculated using the average cell number. The margin of error of the cell counts of each sample was <20%.