Axioskop 2 plus

To test the pollen viability, mature florets before flowering were fixed in 70% (v/v) ethanol and then anthers from the florets were dissected and stained with 1% (w/v) I₂‐KI on a glass slide. The number of the viable pollen grains (deeply stained and round shaped) and inviable pollen grains (lightly stained, small and shrivelled) was counted under a bright field microscope (Axioskop 2 Plus; Zeiss). Three shots were selected for each slide for the statistics of I₂‐KI staining rate of pollen grains.
For histological analysis, anthers of different developmental stages were fixed in 4% paraformaldehyde in 0.025 M sodium phosphate buffer (pH 6.8) overnight at 4 °C. The samples were washed in PBS and dehydrated by washing them through conventional ethanol series of 30 min each and embedded in HISTORESIN (LEICA, cat#702218500) according to the manufacturer’s instructions. 4‐μm sections were cut with a Leica microtome and stained with 0.1% toluidine blue, then observed and photographed with Zeiss Axioskop 2 Plus fluorescence microscope.

The protein fibril formation was checked by fluorescence microscope (Axioskop 2 plus, Ziess, Germany. First, the proteins were dissolved in buffer A at a concentration of 2 mg/mL and incubated at 60 °C in the presence of 1 M GdnHCl for 4 days. After the incubation period, protein samples were prepared at a concentration of 0.15 mg/mL and incubated with 20 μM ThT for 5 min. Then, the green filter (GPF) was used with an excitation wavelength of 469 nm and an emission wavelength of 525 nm to observe the protein fibrils under the fluorescence microscope^{17 (link)}.

Fluorescent staining ethidium bromide (EB)/acridine orange (AO) (Sigma-Aldrich) was performed to assess rates of cellular viability (Live/Dead). Initially cells were seeded in 6 well (monolayer) and 24 well (spheroids) cell culture plates and treated with IC50 concentration of ZEO for 24 h. then cells and spheroids washed with PBS, after which, a solution containing EB/AO was added, the stained cells were immediately visualized and imaged under an fluorescence microscope (Axioskop 2 plus, Ziess, Germany). The differentiation between viable, apoptotic, and necrotic cells is based on the difference between dye permeability into intact cell membrane. Green cells represent viable cells and are stained only with AO, green and orange cells with condensed chromatin represent early and late apoptotic cells and are stained with both AO and EB (with moderate alteration in membrane permeability), finally necrotic cells are orang and stained with EB. Ten photos were taken of randomly selected areas of the stained slides to ensure that the data obtained were representative.