Cfi planapo lamda objective

Leaves from four-week-old plants were fixed in 3.7% formaldehyde in cold Tris buffer (10 mM Tris-HCl pH 7.5, 10 mM NaEDTA, 100 mM NaCl) for 20 minutes. Formaldehyde solution was removed, and the leaves were washed twice for 10 minutes in Tris buffer. The leaves were then finely chopped with a razor blade in 500 μl LB01 buffer (15 mM Tris-HCl pH 7.5, 2 mM NaEDTA, 0.5 mM spermine-4HCl, 80 mM KCl, 20 mM NaCl, and 0.1% Triton X-100). The lysate was filtered through a 30 μm mesh (Sysmex Partec, Gorlitz, Germany). 5 μl of lysate was added to 10 μl of sorting buffer (100 mM Tris-HCl pH 7.5, 50 mM KCl, 2mM MgCl₂, 0.05% Tween-20, and 5% sucrose) and spread onto a coverslip until dried. Cold methanol was added onto each coverslip for 3 minutes, and then rehydrated with TBS-Tx (20 mM Tris pH 7.5, 100 mM NaCl, 0.1% Triton X-100) for 5 minutes. The coverslips were mounted onto slides with Vectashield mounting medium DAPI (Vector Laboratories, Burlingame, CA). Nuclei were imaged under a Nikon Eclipse Ni-E microscope with a 100X CFI PlanApo Lamda objective (Nikon, Minato City, Tokyo, Japan). Digital images were obtained using an Andor Clara camera. Z-series optical sections of each nucleus were obtained at 0.3 μm steps. Images were deconvolved by ImageJ using the deconvolution plugin.