Hc pl apo cs2 20x 0.75 dry objective

Seeds were sown on plates containing half-strength MS medium with 0.6% (w/v) agar and grown vertically for 5 days before transfer to liquid half-strength MS medium without (mock) or with 20 µM zebularine, 20 nM CPT or 10 µM ICRF-187 for 24 h. Afterwards, the seedlings were placed in 10 mg mL^–1 propidium iodide (PI, Sigma) on slides and immediately analyzed and photographed using a Leica confocal microscope TCS SP8 (Leica, Wetzlar, Germany) and HC PL APO CS2 20x/0.75 DRY objective equipped by Leica LAS-X software with Leica Lightning module laser scanning confocal microscope (Leica). The pattern was checked in at least ten individual seedlings per treatment.

Sterilized and stratified seeds were grown vertically on plates with ½ MS medium with 0.8% agar (w/v) for five days and then transferred into liquid ½ MS medium for a 24 h treatment. Mock samples were grown in pure liquid ½ MS medium, while treated plants had medium supplemented with 10 μM MMC. Following the treatment, seedlings were stained with 10 mg.mL^-1 propidium iodide solution (Sigma) on glass microscope slides. Visualization and photography were performed using Leica confocal microscope TCS SP8 (Leica, Wetzlar, Germany) and HC PL APO CS2 20x/0.75 DRY objective equipped with Leica LAS-X software with Leica Lightning module laser scanning confocal microscope (Leica). At least 13 plants for each group were analyzed. The means of the three replicates are depicted. Statistical significance was tested withKruskall-Wallis H-test with post hoc Conover-Iman test of multiple comparisons using rank sums with Benjamini-Hochberg procedure in R 4.2.1 (R Core Team, 2018 ).

Cerebellar sections mounted in Vectashield mounting medium (Vector laboratories) were viewed and imaged using a Leica SP8 a laser scanning confocal microscope (DMI8 CS) with HC PL APO CS2 20x/0.75 DRY objective lenses and controlled by LAS–X software (3.7.2). For each vermal slice, the area imaged included the inner portion of visually identified lobules V/VI and lobules IX/X (see Fig. 6b). Within a set of experiments, all behavioral, staining and imaging procedures were performed in parallel. All images in this study were acquired sequentially with identical settings. For each cerebellar section, a stack of 3–5 focal images near the highest fluorescence level were taken at 5 μm intervals. Mean fluorescence intensity in the granule cell layer or the molecular layer of the cerebellar cortex was quantified using ImageJ software (version 1.53c). The level of fluorescence at the focal plane with the highest mean fluorescence intensity is presented in Figs. 6 and  10, where representative images are displayed with the same contrast and brightness settings. Imaging and analysis were conducted by an experimenter who was blind to the experimental condition.