Model bx41

The percentages of red deer spermatozoa with viable plasma membranes and apoptotic-like changes in plasma membranes were determined with the Vybrant Apoptosis Assay Kit #4 (Molecular Probes Inc., Eugene, OR, USA) according to a previously described method [22 (link)] with some modifications. First, to visualize all cells, 1 µL of JC-1 was added to 200 µL of the sperm suspension (10 × 10⁶ sperm/mL) and incubated for 10 min at 37 °C. Then, 2 μL of YO-PRO-1 solution (100 μM) and 2 μL of PI (2 μM) were added to the stained sample and incubated for 5 min at 37 °C. After incubation, stained sperm cells were examined under a fluorescence microscope (Olympus, model BX41, Tokyo, Japan) at 600× magnification. A minimum of 200 cells per slide were examined in each aliquot. Four populations of spermatozoa were identified: with an unstained head, but with a visible mid-piece (viable sperm without apoptotic-like changes; YO-PRO-1⁻/PI⁻); with a green head (sperm with apoptotic-like changes in the plasma membrane; YO-PRO-1⁺/PI⁻), with a red and green head (moribund/dying sperm cells with dual fluorescence; YO-PRO-1⁺/PI⁺), and sperm with a red head (dead/necrotic cells; YO-PRO-1⁻/PI⁺).

The biofilm coated anodes were cut and transferred into 50 ml phosphate buffered saline (PBS) solution^{66 (link)}. Raman spectral analysis was carried out using micro-Raman spectrometer at a wavelength of 488 nm. Argon-Krypton mixed ion gas laser was used as a light source for excitation. The spectrometer was equipped with an optical microscope (Model BX 41, Olympus, Japan), single monochromator (Model T64000, Jobin Yvon Horiba, France), an edge filter and a Peltier-cooled CCD (1024 × 256 pixel, Model Synpse-1024X, Jobin Yvon, Horiba, France) detector. The spectral acquisition was obtained at an integration time of 0.2 s to obtain high-contrast resonance spectra for c-type cytochromes.

L. donovani promastigotes in late log phase (5×10⁶ cells/ml) were labelled with 500 nM of mitochondrion specific dye, Mitotracker Red (Invitrogen) [46] (link) in serum free M199 media and incubated for 30 mins in BOD at 24.0±1°C. The culture was harvested, washed twice with PBS, and fixed in 1.0% formaldehyde for 30 mins at RT and cells permeabilized with 0.1% Triton X-100 for 10 mins. After that, 0.1 M glycine solution was added and incubated for 10 mins at RT, centrifuged at 3000×g at RT and cell pellet resuspended in 200 µl PBS. Finally, 40 µl of the cell suspension was spread on microscopic slide and dried completely at RT. The slide was blocked with TB buffer (PBS with 0.1% Triton X-100 and 0.1% BSA). The fixed parasites were incubated with anti-LdTryS antibody diluted in TB buffer (1∶500) for 1 hr at RT and secondary antibody FITC-conjugated goat anti-rabbit IgG at 1∶2000 dilution (Santa Cruz) for 1 hr at RT. Cells were washed twice with PBS and labelled with 0.01 µg/ml DAPI (Sigma) in TB buffer for 15 mins, RT. The cells were washed thrice and immunofluorescence was observed in microscope (Model BX 41 Olympus).

Vesicle size of different niosomal formulations were observed under an optical microscope (Olympus Model BX 41, Japan) at suitable magnification. The measurements were done in triplicate and vesicle size was recorded. The zeta potential of the prepared niosomal formulations was determined by Zetasizer Nano ZS-90 (Malvern Instruments Ltd., UK) using 0.1 M KCl buffer in demineralized water at 25 °C (Levchenko, 2002 (link)).

For histological examinations, liver, pancreas and white adipose tissue (WAT) from three animals per group were isolated on day 32 after treatment of compounds. The tissue samples were fixed in formalin solution (10%) for one week at room temperature, dehydrated by graded ethanol, cleared using graded xylene, and embedded in paraffin wax. 5 μm thick sections were cut using rotary microtome (RM2245, Leica Microsystems GmbH, Wetzlar, Germany), fixed on glass slides, stained with eosin and hematoxylin, and observed using a microscope (Model BX41, Olympus Corporation, Tokyo, Japan). For Langerhans cells, the average areas of six islets per specimen were measured using ImageJ software (version 1.48, National Institute of Health, MD, USA). Area was expressed as μm². The quantification of adipocytes were performed as reported [25 (link)], using adipocyte quantification tool, where area was measured in μm².