Planapon60 osc

Live-cell imaging analysis was performed using an Olympus inverted microscope IX70 equipped with a CoolSNAP HQ² charge-coupled device as previously described (Photometrics, Tucson, AZ, USA; [27 (link)]). Briefly, a touch of the sporulating cells on the ME plate was resuspended in the medium of EMM minus nitrogen with the supplements. The solution was added to a lectin-coated (0.2 mg/mL; lectin from Glycine max, Sigma-Aldrich, Tokyo, Japan) glass-bottom dish (MatTek Corp, Ashland, OR, USA). The cells were allowed to adhere to the glass slide via gravity for 5 min. The cells were observed using the Olympus oil-immersion 60× objective lens (PlanApoN60× OSC; NA = 1.4; Olympus, Tokyo, Japan). Optical section images were acquired at 0.5-μm intervals every 6 min using DeltaVision softWoRx 5.5 (Applied Precision, Inc., Seattle, WA, USA). The images were enhanced by three-dimensional constrained iterative deconvolution in softWoRx 5.5.

Tetrahymena cells expressing GFP-tagged Nups were first fixed with cold methanol for 20 min, and then additionally fixed with 4% formaldehyde in PBS for 20 min. After treatment with 1% bovine serum albumin (BSA), cells were treated with 5 µg/ml anti-GLFG monoclonal antibody 21A10 for 2–3 h (Iwamoto et al., 2013 (link)). After washing with PBS, cells were treated with Alexa Fluor 594-conjugated goat anti-mouse IgG at 1:1000 dilution for 1 h (Thermo Fisher Scientific). Images of 40 z-sections with a 0.2-μm interval were taken for cells by using the DeltaVision microscope system with an oil immersion objective lens PlanApoN60OSC (NA=1.4) (Olympus), and were processed by deconvolution using SoftWoRx software equipped with the microscope.