Lsm image browser software

For nuclear layer staining, we embedded the eyecups in OCT embedding medium (n = 5). Eyecups were sectioned along the vertical meridian on a cryostat at a thickness of 10 μm. TOPRO-3 (Invitrogen, Carlsbad, CA; T3605, dilution 1:1,000) was incubated for 10 minutes and washed for 30 minutes with 0.1 M PB and cover-slipped with Vectashield mounting medium. The Zeiss LSM image browser software was used to measure the thickness of ONL. The measurements were taken within 2 mm from the optic nerve.

The aortas were prepared en face and stained Oil Red O (Sigma-Aldrich, St. Louis, MO, USA). The aortic lesion area and total aortic area were calculated using LSM Image Browser software.
The hearts with the ascending aorta were embedded in OCT compound (CellPath, Newtown, UK), snap frozen and sectioned (10 μm thickness) for histological and immunohistochemical analysis, according to the standardized cross-section protocol, as described before [51 (link),52 (link)]. To evaluate the lesion area and plaque collagen content, nine sections per animal were stained Oil Red O (Sigma-Aldrich, St. Louis, MO, USA). Immunohistochemistry was performed with antibodies against CD68 (dilution 1:800; Serotec, Kidlington, UK) and smooth muscle α-actin (SMA) (dilution 1:800; Sigma-Aldrich, St. Louis, MO, USA). In situ zymography was performed to demonstrate non-specific activity of gelatinases using the standard protocol [53 (link)]. All section images were captured using an Olympus Camedia DP71 digital camera and analyzed using LSM Image Browser software (Zeiss, Jena, Germany).

For frozen sections, mice were anaesthetized, and pancreata were rapidly dissected, fixed in 4% paraformaldehyde, immersed in 20% sucrose before freezing and then sectioned at a thickness of 7 μm. After antigen unmasking, the slides were blocked with 5% BSA/PBS and incubated at 4°C, with guinea pig anti‐insulin (1:500; Millipore), rabbit anti‐GCK (1:100; Santa Cruz) and rabbit anti‐GLUT2 (1:200; Santa Cruz), followed by secondary antibodies (Invitrogen). Slides were viewed under an LSM‐710 confocal microscope (Carl Zeiss). Using LSM Image Browser software (Carl Zeiss), multiple sections from three mice per genotype, separated by at least 200 μm from each section, were assessed for quantification of GCK and GLUT2.

Immunofluoresence microscopy was performed using confocal microscopy (LSM510 META; Carl Zeiss) equipped with a microscope (Axiovert 200 M; Carl Zeiss), a plan Apochromat × 100/1.4 NA oil immersion lens and LSM image Browser software (Carl Zeiss) as described29 (link) with slight modifications. The list of antibodies with source and conditions of indirect immunofluorescence is shown in Supplementary Table 2. In some immunofluorescence experiments, we used Can Get Signal immunostain Solution A (TOYOBO). For induction of primary cilia, RPE1 and IMR-90 cells were seeded on sterile coverslips, grown for 1 day and then subjected to serum starvation. For detection of primary cilia, cells were placed on ice for 20–30 min before fixation with cold methanol, and stained with anti-acetylated tubulin antibody29 (link). BrdU incorporation was evaluated using DNA Replication Assay Kit (Millipore). Quantification of fluorescence intensity was performed using ImageJ 1.45r software.

Approximately 1X10⁶ spores were equally distributed in 50μl droplets on thinly-poured, fully-supplemented solid minimal media agar. Flame-sterilized coverslips were placed on top of the spore droplets. Petri plates were incubated at 30°C until desired developmental stage was reached. The area of agar directly underneath the coverslips was dissected using a sterile scalpel and mounted onto flame-sterilized microscope slides. Each slide was viewed under 100X oil immersion objective lens on a Zeiss LSM 510 VIS/META confocal microscope at room temperature, with an upright AXIO Imager M1 microscope stand and Diode (405 nm), Argon (458, 477, 488, 514 nm), HeNe1 (543), HeNe2 (594) and HeNe3 (633 nm) laser lines. Using bleach settings on LSM Imaging software, a ROI was designated to highlight a particular nucleus, and photo-conversion was conducted at: 100–150 iterations, 2.56μsec pixel dwell time, at 75–100% 405nm laser power. Z-stack images were captured before conversion, after conversion, and specific time-intervals between each image. Brightness increased for entire panel images for Dendra2 experiments to improve visualization wherever necessary. Maximum intensity projections of z-stack images and image settings compiled and adjusted respectively in Image J 1.48v (Java 1.6.0_20 (64-bit)) and Zeiss LSM Image Browser software (Version 4,2,0,121).

The quantification of the number of tubercles was performed for TV (the most active sample in tegument) using a confocal microscope. After the established times or in the occurrence of death, the parasites were fixed in formalin-acetic-alcohol solution (FAA) and analyzed under a confocal microscope (Laser Scanning Microscopy, LSM 510 META, Zeiss) at 488 nm (exciting) and 505 nm (emission) as described by [28 (link), 29 (link)]. A minimum of three areas of the tegument of each parasite were assessed. The numbers of tubercles were counted in 20,000 μm² of area calculated with the Zeiss LSM Image Browser software. A blind analysis was performed by observer with experience and training in parasitology.