Lsm 880 axioobserver confocal microscope

All slides were analyzed using a Zeiss Axio Imager.M2 microscope equipped with an Axiovision digital imaging system (Zeiss) using 20× or 40× objectives or a Zeiss LSM 880 Axio observer confocal microscope with Airyscan using a 63× objective (Zeiss). The 20× objective for the Axio imager was a Plan-apochromat with a numerical aperture of 0.8 M27. The 40× objective for the Axio imager was a Plan-apochromat with a numerical aperture of 0.95 Korr M27. The 63× objective for the Zeiss LSM 880 confocal microscope was a Plan-apochromat oil-immersion objective for Airyscan with a numerical aperture of 1.4. Fluorescent donkey secondary antibodies were used that were conjugated to Alexa Fluor 488, Cy3, Cy5, or Alexa Fluor 796. Imaging of fixed tissue was performed at room temperature; all specimens were mounted in ProLong Gold antifade reagent before imaging. The camera used to acquire images on the Axio Imager microscope was the AxioCam HRm Rev.3. For images acquired using the Zeiss LSM 880 Airyscan, superresolution processing was used. Axiovision digital imaging system and Zen blue software were used to export single-channel tiff images for all images acquired. Adobe Photoshop was used to merge images.

Fixed samples were mounted on glass coverslips using Fluoromount-G and imaged using a confocal, widefield, or high-resolution Airyscan confocal microscopy as indicated in the figure legends. Widefield images were acquired using an Olympus IX-83 microscope using UPlanSAPO 40× silicon oil (NA 1.25; Fig. 2 A and Fig. S4 B) or PlanApo N 60× TIRF microscope objective (oil, NA 1.45; Fig. S2 C). Confocal images were acquired using a Zeiss LSM 710 confocal microscope with 63× Plan Apochromat oil objective (NA 1.4; Fig. 1 A and Fig. S1, D and E), Zeiss LSM 710 META with 63× Alpha Plan Apochromat oil objective (NA 1.46; Fig. 3, D and F), or Zeiss LSM880 Axio Observer with 63× Plan Apochromat oil objective (NA 1.4; Fig. 2 F and Fig. S2, A, B, D, and E). Airyscan confocal imaging was performed using Zeiss LSM 880 AxioObserver confocal microscope with Airyscan detector and 63× Plan Apochromat oil objective (NA 1.4). Images were acquired in superresolution scan mode (Fig. 1, B and E; and Fig. 5, A and B).