Fluorescent microscope

The assessment of sperm viability was performed using a kit supplied by Halotech DNA S.L., a Madrid-based firm in Spain. The kits include acridine orange (AO) and propidium iodide (PI). AO is a cell-permeant DNA-binding dye that stains both live and dead cells with green fluorescence, while PI is a membrane-impermeant DNA-binding dye that stains dead cells red with damaged membranes. At the outset, the frozen was diluted to a concentration of 10–15 × 10⁶ sperm/ml. Following this, a quantity of 10 μL of cryopreserved semen was mixed with a solution containing 1.0 μL of AO and PI. Subsequently, the combination was examined using a fluorescent microscope produced by Optika Srl, Italy. The live sperm had green fluorescence as a consequence of AO retention, but the damaged sperm exhibited red fluorescence owing to PI penetration. In each case, an evaluation was conducted on a sample size of 300 spermatozoa.

The cell death in Drosophila brain was analyzed as per the method described by Mitchell and Staveley [17 ]. Flies (5 flies/treatment; 5 replicates/group) were placed in 70% ethanol in a 2 mL microcentrifuge tube for a minute. The brains were isolated in Ringer's solution under stereo zoom microscope. After removing the Ringer's solution about 100 μL of freshly prepared acridine orange (5 μg/mL) was added for 5 minutes. The brain was rinsed by Ringer's solution, immediately viewed, and photographed through fluorescent microscope (Optika, Italy). The image analysis program Image J (available online at http://rsb.info.nih.gov/ij/) was used to analyze the gray scale values for each brain.

The cell death in the Drosophila brain was analyzed as per the method described by Mitchell and Staveley⁶⁷ . After 24 days of exposure of PD flies to various doses of kaempferol, the brain were isolated in Ringer’s solution under stereozoom microscope. Flies (20/treatment; 5 replicates/group) were placed in 70% ethanol in a 2 ml microcentrifuge tube for a minute. After removing the Ringer’s solution, about 100 µl of freshly prepared acridine orange (5 µg/ml) was added for 5 min. Each brain was rinsed by Ringer’s solution, immediately viewed and photographed through fluorescent microscope (OPTIKA, Italy). The image analysis program Image J (available online at https://rsb.info.nih.gov/ij/) was used to analyze the grey scale values for each brain.