The cells were fixed with 4% paraformaldehyde for 30 min and permeabilized in 0.1% Triton X-100 in PBS for 30 min. These studies were conducted similarly as described using specific antibodies as indicated (20). Alexa Fluor 488– and/or Alexa Fluor 568–conjugated secondary antibodies (Invitrogen) were used. The nuclei were stained with the DNA-intercalating dye 4′,6-diamidino-2-phenylindole. The cells were analyzed under a fluorescence microscope (Olympus IX71 FluoView). Images were captured using the DP72 Olympus camera system and cellSens software. Alternatively, images were captured using Zeiss Axio Imager.A2 (white field) or Zeiss LSM 880 Airyscan (confocal) together with the Zen software. The average arbitrary JLP focus intensity per cell was quantitated using ImageJ. High-resolution STED images were acquired on a Leica TCS SP8 STED 3X. Cells were labeled with FITC-conjugated α-tubulin antibody and Nup358 antibody with secondary antibody Alexa Fluor 647. Images were acquired at 400 Hz with gated HyD detectors (adjusted between 0.3 and 6 ns) and a 100× oil immersion objective (Leica) with 5× zoom to obtain the desired pixel size (20 nm). Alexa Fluor 647 was excited with the 653-nm line (4 to 10%), and the pulsed 775-nm depletion line (8%) was used to generate STED. FITC was excited with the 495-nm line (1 to 4%) and the continuous-wave 594-nm depletion laser (20%) was used to generate STED. The images were analyzed by Leica Suite X.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.