Axio imager z1 apotome microscope

Immunofluorescence was performed as recently described^{29 (link)}. Cells were fixed with 3% formaldehyde/PBS for 30 min, permeabilized with 0.5% Triton X-100/PBS for 10 min and blocked with 3% BSA/PBS for 1 h. Staining of cleaved caspase 3 (Cell Signaling, #9664) was carried out overnight at 4 °C and with anti-rabbit secondary antibody for 2 h at room temperature. After several washes with PBS, samples were covered with Vectashield/DAPI mounting medium (Vector Labs). Images were acquired using an AxioImager.Z1/ApoTome microscope (Zeiss).

Cells were deposited on poly-lysine coated slides (J2800AMNZ, Thermo Scientific) using a Cytospin 4 centrifuge (Thermo Scientific) at 600 rpm for 10 minutes. Soluble cell fraction was pre-extracted by incubation with cold cytoskeleton buffer (CSK: 10 mM PIPES, pH 7, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 0.7% Triton X-100) (2 x 3 minutes), fixed with 4% PFA-PBS, and saturated with 3% BSA-PBS for 1h at RT. Anti-BLM antibodies (ab476, Abcam; sc-365753, Santa Cruz) were diluted at 1:200 and anti-nucleolin (ab22758, Abcam) was diluted at 1:1000 in saturation buffer and incubated on slides in a humid chamber for 90 minutes. Slides were washed 3 x 5 minutes with PBS-0.01% Tween, incubated protected from light in a humid chamber with secondary antibody (A11008, Invitrogen) 1:500 for 45 minutes at RT. Washed again 3 x 5 minutes with PBS-0.01% Tween, incubated with DAPI (20 μg/ml) in H₂O for 5 minutes and washed 3 times with H₂O. Slides were air dried and mounted with Prolong Gold (P36930, Invitrogen) and let to dry overnight. Image acquisition was performed with a ZEISS Axio Imager Z1 Apotome microscope and analysis was done with Omero server.

Live larvae were imaged for mRFP fluorescence on an Axio ImagerZ.1 ApoTome microscope (Zeiss) with AxioVision software (Zeiss). Z-stack images were flattened using the AxioVision Multi-Image Projection (MIP) tool. SCORE imaging techniques [115 (link)] were used to hold live larvae and optimized for high-quality fluorescent imaging as follows: live larvae were transferred into 2.5–2.6% methylcellulose and drawn into borosilicate glass capillaries (World Precision Instruments WPI #1B120-3). 80% glycerol was used as the imaging medium between capillary and cover slip. Brightfield images of live larvae were obtained either by mounting the specimen in 1.5% methylcellulose and imaging with a Zeiss Axiophot microscope using the Zeiss AxionCam HRc camera and AxioVision software, or by SCORE imaging on a Zeiss Axioplan2 microscope using a Canon PowerShot A640 camera with Remote Capture Task and Canon Image Browser software.

hTERT-RPE1 cells were grown on coverslips, fixed for 20 minutes at room temperature (RT) in 4% paraformaldehyde in PBS, permeabilized for 5 minutes at RT in 0.3% Triton X-100 in PBS and saturated for 1 hour RT in normal goat serum 10%, BSA 1% in PBS. Cells were stained overnight at 4 °C with anti-ZO-1 primary antibody (610966, BD Transduction Laboratories, California, USA). After washes, cells were incubated for 1 hour at RT with anti-mouse secondary antibody conjugated to Alexa Fluor 488 (A-11029, Thermo Fisher Scientific, Massachusetts, USA). Cells were stained with DAPI for 5 minutes and coverslips were mounted with Fluoromount-G solution (17984-25, Electron Microscopy Sciences, Pennsylvania, USA). Images were obtained using Z-stacks on a Zeiss Axio Imager Z1 ApoTome Microscope. Length of ZO-1 labeled fragments was measured with an ImageJ adjusted customized macro from Borras et al.^{21 (link)}. Enhancement of linear structures was obtained by computation of the smallest eigenvalue of Hessian tensor with the ImageJ plugin FeatureJ ((Erik Meijering, Erasmus University Medical Center, Rotterdam, Netherlands. Plugin available from https://imagescience.org/meijering/software/featurej). Obtained images were binarized and skeletonized. Skeleton were analyzed and the average branch length distribution was compared between siNeg and siCFH.

Transgenic animals expressing flp-1p(1.5 kb):GFP, flp-1p(513 bp):FLP-1(cDNA):mCherry, or tdc-1p::GFP were placed on full-lawn PA14 plates to eliminate variations due to avoidance behavior. Transgenic animals grown on E. coli OP50 plates were randomly transferred to a full-lawn PA14 plate or a control OP50 plate, and incubated for 4 or 10 h at 20°C before imaging.
Live animals were mounted on 2% agarose pads containing 5 mM sodium azide. Fluorescence was collected with a 40X objective on a Zeiss Axio Imager.Z1 Apotome microscope with a Zeiss AxioCam MRm CCD camera (Figures 3E and 3F, 3G, 5E, 5F, and S2F), or a Zeiss Inverted Axio Observer Z1 LSM 780 laser scanning confocal microscope with a 40x objective (Figures 3C and 3D). Imaging settings were constant across all experiments. Images were processed using ImageJ (NIH, Bethesda, USA) to generate a maximum-intensity Z-projection. Quantification of fluorescence levels was performed by drawing a region of interest (ROI) around the cell body and measuring mean gray scale values.