Lsm image browser version 4

The recorded confocal data were processed using Zeiss LSM Image Browser Version 4.2.0.121 and/or Zeiss ZEN 2009 (Carl Zeiss MicroImaging, Jena, Germany). FLIM data were analyzed by SPCImage Version 5.0 (Becker and Hickl, Berlin, Germany) and MicroSpace (ILC CVTISR Bratislava, Slovakia), which used the NLOPT numerical library (non-linear optimization) for FLIM data analysis [25 ]. The statistical analysis was carried out by the IBM SPSS version 21 (SPSS Inc., Chicago, IL, USA). Results are presented as means ± SD.
To test the normality of the group distribution, the Shapiro–Wilk test was used. The statistical significance of differences was tested by the independent two-population Student’s t-test for normally distributed data and the Wilcoxon’s test for not normally distributed data for a non-parametric analysis between group pairs. A p-value < 0.05 was considered statistically significant.

TF-1 cells were collected on histogrip (Invitrogen, Frederick, MD, USA) coated slides by cytospin (Shandon Cytospin 3, Pittsburgh, PA, USA), were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilized in 0.1% Triton X-100 in PBS for 10 min at RT, followed by blocking with 10% donkey serum (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at RT. Primary antibodies were applied at the manufacturer's recommended concentrations, followed by overnight incubation in a humidified chamber at 4 °C. Fluorophore-conjugated secondary antibodies were applied at the dilution of 1:500 and incubated for 2 h at RT. The cells were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, Eugene, OR, USA) for nuclear counterstaining. Images were captured at a resolution of 2048 × 2048 using a Carl Zeiss LSM 780 (Carl Zeiss, Jena, Germany) confocal microscope with a × 40 Plan objective lens. The LSM Image Browser version 4.2.0.121 (Carl Zeiss MicroImaging, Jena, Germany) was used to analyze the microscopic slides.

Biopsy fragments were obtained by cryosection and prepared for immunofluorescence microscopy as described [28 (link)]. Briefly, slides were rehydrated with PBS (pH 7.4) and unspecific epitopes were blocked with rabbit serum for two hours of incubation. Then, the sections were incubated with mouse anti-human NE (Calbiochem—EMD Millipore Co, Billerica, MA, USA)), anti-human DNA-histone complex (Millipore), anti-CD68 macrophages (Dako) and anti-L. braziliensis polyclonal rabbit serum, as primary antibodies. Anti-rabbit FITC-conjugated (Vector Labs), anti-mouse Alexa Fluor-546 (Calbiochem) and anti-mouse PE-conjugated were used as secondary antibodies. After staining, sections were mounted in Vectashield mounting medium (Vector Labs). Some slides were also mounted on medium containing DAPI (Fluoromount-G, eBioscience). Immunofluorescence analysis was done in a Nikon Eclipse E400 Microscopy (Nikon digital camera DXM1200F and Nikon ACT-1 software, Nikon, Japan). Confocal analysis were done in a Zeiss LSM 510 META confocal microscope (Program for Technological Development in Tools for Health—PDTIS-FIOCRUZ) using excitation at 488 nm or 546 nm and emission at 505–530 nm (band pass system) and 560 nm (long pass system). The images were processed using the program LSM Image Browser—Version 4.2.0.121 (Zeiss, Jena, Germany).

The CNS and attached eye-antennal imaginal discs were dissected from third instar larvae. For direct GFP detection (without GFP immunostaining) and antibody staining, tissues were fixed in phosphate-buffered saline (PBS) containing 4% formaldehyde (Merck), and then washed 3 times (5 min each) with 0.5% Triton X-100 in PBS. The primary monoclonal rat anti-Elav (DSHB #7E8A10) and monoclonal mouse anti-Repo (DSHB #8D12) antibodies were used at concentration 1 μg/ml. They were detected by goat anti-rat IgG antibodies conjugated to AlexaFluor568 (1:800; Invitrogen #A-11077) and by goat anti-mouse IgG antibodies conjugated to AlexaFluor568 (1:800; Invitrogen #A-11031). Finally, tissues were stained with 0.4 μg/ml DAPI dissolved in PBS. All samples were imaged at the same settings using confocal microscope LSM 710 (Carl Zeiss) with 10×/0.45 plan-apo and 20×/0.8 plan-apo lenses. Optical sections were combined using the LSM Image Browser version 4.2 software (Carl Zeiss).

Dissected ovaries were mounted on glass slides in mounting medium (50% glycerol, 0.82 mM KH₂PO₄, 2.6 mM NaH₂PO₄, 75 mM NaCl). Immunostainings were performed as described previously [57 (link)]. The primary antibodies were mouse anti-Fasciclin III (anti-Fas III; 5 µg/mL; #7G10; Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA), mouse anti-beta-galactosidase (anti-β-gal; 5 µg/mL; #40-1a; DSHB) and rabbit anti-GAGA (1:500; kindly provided by Prof. Vincenzo Pirrotta). The secondary antibodies were goat anti-mouse conjugated to Alexa Fluor 488 (1:500; #A-11001; Invitrogen, Carlsbad, CA, USA), goat anti-mouse Alexa Fluor 568 (1:500; #A-11031; Invitrogen) and goat anti-rabbit Alexa Fluor 488 (1:500; #A-11034; Invitrogen). TRITC-labeled phalloidin (1:100; #P1951; Sigma-Aldrich, Saint Louis, MO, USA) was used to visualize F-actin as described previously [58 (link)]. DAPI was used at 0.4 µg/mL to stain nuclei. Samples were imaged using an Axio Observer Z1 (Carl Zeiss, Oberkochen, Germany) and confocal microscope LSM 710 (Carl Zeiss). Optical sections were combined using the LSM Image Browser version 4.2 software (Carl Zeiss).

The interactions between PKH26 + OCCs and GFP + E4 + ECs in angiospheres were imaged using a Zeiss confocal Laser Scanning Microscope 710 (Carl Zeiss). Post-acquisition image analysis was performed with Zeiss LSM Image Browser Version 4.2.0.121 (Carl Zeiss). Spheres were imaged live using glass-bottom microwell plates (MatTek Corporation, Ashland, MA, USA) [65 (link)].

ECs treated with MPs as detailed in specific experiments were fixed in 3.7% formaldehyde. Slides were mounted in a mounting media SlowFade® Gold Antifade Reagent (Invitrogen). Fluorescence Imaging was performed using a Zeiss confocal Laser Scanning Microscope 710 (Carl Zeiss). Post-acquisition image analysis was performed with Zeiss LSM Image Browser Version 4.2.0.121 (Carl Zeiss).