Application suite software

For confocal laser scanning microscopy (CLSM) analysis, cells were seeded on poly-l-lysine–coated ø 18-mm coverslips, fixed with 4% PFA (20 min), permeabilized with 0.1% Triton X-100 (10 min), and blocked with PBS containing 10% FCS (1 h). The slides were incubated with CD63 primary antibody for 30 min at RT followed by incubation with anti–mouse Alexa Fluor 594 secondary antibody for 30 min at RT, mounted with the Vectashield reagent (Vector Laboratories), and sealed with nail polish. CLSM was performed at RT on a TCS SP8 X microscope (Leica Microsystems). Samples were irradiated with a pulsed white light laser at 488 or 594 nm. The signals were detected using a gated Hybrid Detector (HyD; Leica Microsystems). CLSM images were acquired using an oil objective with 63× magnification and 1.4 NA. Application Suite software (Leica Microsystems) was used for image acquisition. ImageJ (National Institutes of Health) software was used for statistical analysis, including calculation of Pearson’s correlation coefficients.

To evaluate possible immune responses in the heart, hematoxylin-and-eosin staining was performed in sections obtained from the infarct (n = 3 slides per heart), the injection sites (n = 6 slides per heart), and remote LV myocardium (n = 3 slides per heart). Sections were analyzed by two independent pathologists blinded to treatment allocation. Analysis was graded according to the International Society for Heart & Lung Transplantation (ISHLT) guidelines, a system used in clinical practice to grade cardiac allograft rejection [39] . Additional histopathology features were assessed, including amount of calcification, granulation tissue, and necrosis. To measure myocyte cross-sectional area, sections obtained from the infarct border zone underwent immunostaining for troponin I (Abcam, Cambridge, MA) and wheat-germ agglutinin (Molecular Probes, Grand Island, NY) (to visualize cell borders); Alexa Fluor conjugated secondary antibodies (Molecular Probes) were used and counterstaining with DAPI (Molecular Probes) was performed. Sections were imaged using a confocal laser scan microscope (Leica Microsystems, Buffalo Grove, IL) and images were processed by Leica Application Suite software.

Testicular tissue sections were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol. Hemalun eosin saffron (HES) staining was performed to visualize seminiferous tubule architecture, as previously described^{54 (link)} and periodic acid Schiff reaction (RAL diagnostic, Martillac, France) was used to detect the pink-labelled acrosome of spermatids and visualize the progression of spermatogenesis. Tissue structural integrity was assessed in each HES-stained testicular tissue section by calculating a global lesional score on a scale from 0 to 10 (0–5 for epithelial integrity and 0–5 for nuclear alteration, with 0 representing the complete absence of alteration and 5 representing the most important damage)^{54 (link)}.
Analyses were conducted with a light microscope (DM4000B, Leica Microsystems GmbH) equipped with a Leica Application Suite software. For each animal, the global lesional score was obtained from 60 cross-sectioned tubules in 2 sections separated by an interval of at least 35 µm.

We performed immunofluorescence as we have previously reported^{19 (link)}. Primary antibodies include anti-SEC24C (Cell Signaling #14676) at 1:100, anti-UBF (Santa Cruz sc-13125) at 1:200, anti-Nucleophosmin/B23 (Abcam ab10530) at 1:500, and anti-Fibrillarin (Cell Signaling # 2639) at 1:400 dilutions. We used the Alexa 555 goat anti-mouse (Cell Signaling #4409) or goat anti-rabbit (Cell Signaling #4413) secondary antibodies at 1:1000 dilution for 1 h at room temperature. We performed aggresome staining using the PROTEOSTAT® Aggresome Detection Kit (Enzo Life Sciences, Farmingdale, NY, USA). Coverslips were mounted using ProLong Gold Antifade mountant with DAPI (Invitrogen). Slides were visualized and images obtained using a laser scanning confocal TCS SP8 microscope controlled by the Leica Application Suite Software (Leica). All images were obtained using a ×63 oil immersion objective and 405-, 488-, and 594-nm lasers.

Aliquots of select aldehyde-preserved samples were sent to the Marine Biology Research Group of Ghent University for nematode morphotype analyses. The >32-μm fraction of sediments was subject to a Ludox^® density-gradient centrifugation; the supernatant was stained with Rose Bengal and metazoan meiofauna were sorted under a stereomicroscope and identified to coarse taxonomic level [72 ]. Nematodes were handpicked and mounted on slides. After, these specimens were examined with a Leica DMR microscope and assigned to genus when possible. Occasionally, areas of interest (e.g. heads, tails) were digitally imaged using the Leica Application Suite software. All males of the most abundant nematode genus were assigned to morphospecies based on morphometric characteristics (e.g. body length, body width, position and size of the amphids). The analyzed samples were from a known volume of sediment, so metazoan densities could be calculated; these data were added to counts obtained via sieving and with Percoll isolations as described above. When loriciferans were encountered in these preparations, images were obtained in some cases.