Hc pl apo cs2 20 0.75 dry objective

In order to assess apoptosis levels in the M1 and CA1 areas, TUNEL histochemistry was performed according to the instructions provided by commercial kit manufacturer (in situ Cell Death Detection, TMR red, Roche; Germany). Animals were anesthetized and perfused through the ascending aorta with a solution of normal saline for ∼3 min, followed by 4% paraformaldehyde in 0.1 M PB for 5 min. Brains were removed and post-fixed in 4% paraformaldehyde at 4 °C overnight and then cut into 30-μM-thick coronal sections including cortex and hippocampus. These frozen sections were incubated in permeable buffer (0.3% Triton X-100 in PBS) for 15 min at room temperature. Then the sections were incubated with TUNEL reaction mixture for 60 min at 37 °C in a chamber with moderate humidity, rinsed with PBS. Sections were incubated with DAPI (1:1000, Cell Signaling 4083S) for 15 min. TUNEL positive cells were acquired on a Leica TCS SP8 confocal microscope equipped with HC PL APO CS2 ×20/0.75 DRY objective at the Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine. Image analysis was performed by ImageJ 1.52a (US National Institutes of Health). The images of different channels were thresholded, cell numbers were determined according to the DAPI channel threshold image.

Seeds were grown on vertically positioned plates with MS medium for 5 days, and then transferred to liquid MS media without (mock) and with 20 μM zebularine, 20 μM AC, or 20 or 10 μM DAC for another 2 days. Subsequently, the seedlings were stained with 10 mg ml^–1 of propidium iodide (PI) solution (Sigma) for 3 min, followed by a rinsing step with sterilized water. Then, they were placed on slides in a drop of water and analyzed using a Zeiss AxioImager Z2 microscope (Zeiss, Oberkochen, Germany) equipped with a high-performance DSD2 confocal module (Andor) and Plan-APOCHROMAT 10×/0.45 objective. Representative phenotypes were imaged with a Leica confocal microscope TCS SP8 (Leica, Wetzlar, Germany) and HC PL APO CS2 20×/0.75 DRY objective equipped by Leica LAS-X software with a Leica Lightning module laser scanning confocal microscope (Leica).