Lsm 5 live microscope

High-speed confocal imaging was performed by time-lapse collection of single optical section at a frame rate of 370fps using a Zeiss LSM 5LIVE microscope. Images were collected from living embryos expressing membrane-GFP driven by MCC-specific promoter and from embryos injected with Ribc2 morpholino.
For filming the MCC intercalation, living embryos of either control or Rfx2 morpholino-injected animals were put on a round cover glass in custom machined dishes (Kieserman et al., 2010 (link)). Embryos were gently pushed down by a small piece of cover glass. Images were collected every 3–5 min and were then processed into a time-lapse movie using Fiji software.
For IFT imaging, embryos expressing GFP-IFT20 alone or with Ttc29 MO were mounted flank down in 0.8% LMP agarose. Single confocal slices were collected at ∼2 fps using an LSM 5LIVE confocal microscope, as previously described in Brooks and Wallingford (2012) (link).

Images were acquired with a Leica TCS SP5-II upright confocal microscope using a 63x oil immersion lens (NA 1.4, HCX PL APO) with or without digital zoom as a series of short Z-stacks. Maximum intensity projection processing of Z-stacks was done in Fiji (ImageJ) software. Mucus layer thickness was measured using the Leica distance measurement tool (LASAF). The width of the inner mucus layer was determined by the average of 4 measurements per field with 4 fields measured per section. Whole tissue images were digitally scanned using the Zeiss Mirax Desk Scanner with 20x/0.8NA objective. Bacterial distance analysis was performed on colon images taken at 63x magnification with a 4x digital zoom by determining the XY coordinates of each bacterial cell in MetaMorph (Molecular Devices) software and measuring the distance from their center. For quantification of bacterial density and invasion into the mucus layer, whole tissue cross-sections were tile scanned in short Z-stacks using an inverted laser scanning confocal Zeiss LSM 5-Live microscope at 63x magnification. For bacterial quantification, a threshold based on the RGB color combination and intensity of each bacterial species was generated with the color thresholding option in MetaMorph. Thresholded objects of 1μm in size were counted as a single bacterial cell with MetaMorph’s integrated morphometric analysis tool.

Muscle cross-sections (6 µm) were fixed for 15 min at room temperature in 4% paraformaldehyde, permeabilized using 1% Triton X-100 for 15 min at 4 °C, and blocked for 2 h at room temperature using 1% normal serum [19] (link), [40] (link). Prepared sections were probed overnight using antibodies to nSMase3 (1:100), ATP synthase-α (1:100), and annexin II (1:50). Secondary antibodies were incubated for 45 min (donkey anti-rabbit DyLight 649; donkey anti-mouse DyLight 594; 1:200). Sections were mounted with ProLong Gold antifade reagent with HEK293 (Invitrogen) and examined by fluorescence microscopy (LSM 5 Live Microscope, Zeiss, Thornwood, NY).