Ixon x3 du897

For immunofluorescence microscopy, cells were fixed in 3% formaldehyde/0.1% Nonidet P-40 in HMEK and allowed to settle onto coated multiwall slides for 2 min followed by submersion in −20°C methanol for ∼4 min. After air-drying, the slides were washed with phosphate-buffered saline (PBS), and blocked 2% bovine serum albumin (BSA) in PBS. The following primary antibodies and dilutions were used: mouse anti-IC2 (1:4) and rabbit anti-ODA16 (1:5). Specimens were incubated overnight at 4°C with primary antibodies in 2% BSA in PBS, and subsequently with secondary antibodies linked to Alexa Fluor 488 or 568 (1:1000; Invitrogen) for 90 min at room temperature. Specimens were mounted with ProlongGold (Invitrogen). Images were captured using a EMCCD camera (Andor iXon X3 DU897) and the Elements software package (Nikon) on a Nikon Eclipse Ti-U inverted microscope equipped with a 40×/0.75 NA objective (Nikon). Image brightness and contrast were adjusted using Photoshop (CC 2017; Adobe) and figures were assembled using Illustrator (CC 2017; Adobe).

For in vivo imaging, a Nikon Eclipse Ti-U inverted microscope equipped with a 60×/1.49 numerical aperture (NA) TIRF objective and a through-the-objective TIRF illumination system was used. Excitation light was provided by 75-mW, 561-nm and 40-mW, 488-nm diode lasers (Spectraphysics), and filtered by a Nikon GFP/mCherry TIRF filter cube (Lechtreck, 2013 , 2016 ). The two-color emission was separated by using an image splitting device (Photometrics DualView2), and documented at 10 frames/s using an EMCCD camera (Andor iXon X3 DU897) and the Elements software package (Nikon). For photobleaching the entire flagellum, the laser intensity of the 488-nm laser was increased to 10% for 4–10 s. For photobleaching a specific area, a focused 488-nm laser beam was passed through the specimen in epifluorescence mode. To prepare the observation chamber, 10 μl of cells was placed on a 24 × 60 mm no. 1.5 coverslip previously applied with a ring of petroleum jelly. The cells were allowed to settle for ∼1–10 min, mixed with an equal volume of 10 mM HEPES and 6.25 mM EGTA (pH 7.4) under a 22 × 22 mm no. 1.5 coverslip. The images were analyzed and kymograms and walking averages were generated in FIJI (= ImageJ; National Institutes of Health). Merged images or kymograms were produced using Photoshop and figures were assembled in Illustrator (Adobe).