Eclipse 80i microscope

For each animal, a series of 30-μm-thick coronal brain slices of the entire brain (240 μm between consecutive slices) was used for NeuN immunolabeling; we used equivalent series among animals. Micrographs were acquired with a Nikon Eclipse 80i microscope, using the NIS-Elements software, under standardized conditions. For each slice, the areas corresponding to the contralateral hemisphere (Contra) and to NeuN⁺ tissue within the ipsilateral hemisphere (Ipsi_NeuN) were encircled using ImageJ. The infarct area (IA) was estimated accounting for edema or tissue shrinkage: IA = Contra − Ipsi_NeuN. Infarct volume was obtained by volumetric integration.

The myocardial sample was fixed with 4% paraformaldehyde for 24 h, dehydrated through increasing concentrations of ethanol, and then embedded in paraffin. LV sections (5 μm) were stained with Masson's trichrome (Sigma-Aldrich) following the manufacturers' instructions. In brief, nuclei are stained with Weigert's iron hematoxylin, and cytoplasm and muscle are then stained with Beibrich scarlet-acid fuchsin. After treatment with phosphotungstic and phosphomolybdic acid, collagen is demonstrated by staining with aniline blue. Rinsing in acetic acid after staining renders the shades of color more delicate and transparent. Images were recorded by A Nikon Eclipse 80i microscope and NIS Elements software. Dedicated software was used to analyze the area of fibrosis in the border zone on Masson's trichrome-stained sections. Percent scar circumference was determined as total infarct circumference divided by total LV circumference × 100.

Our enumeration of microbial and viral abundances followed established techniques (Suttle and Fuhrman, 2010 (link)). Sediment extracts were filtered through 0.02 μm pore size Anodisc filters, stained with 40X SYBR Green I, and enumerated at 1000X magnification using a Nikon Eclipse 80i microscope and NIS-Elements BR imaging software (v3.2). Per sample, between 200 and 500 microbes and VLPs were counted from 1140 fields of 100 μm² each. Given the microscope’s field of view at 1000X magnification, each sample was effectively ‘measured’ 15 times, leading to a relative uncertainty of each abundance measurement of <25% (defined as standard deviation of all measurements divided by the mean of all measurements). Visibly dividing cells (VDC) were also enumerated. Viral counts were back-corrected to in situ concentrations using the storage decay relationships described in Colangelo-Lillis et al. (2016b) (link). Methods employed here did not distinguish between Bacteria, Archaea, and small single-celled Eukaryota. Members of each domain have been reported in both springs (Perreault et al., 2007 (link), 2008 (link); Niederberger et al., 2010 (link); Lay et al., 2012 (link), 2013 (link)) and are herein collectively referred to as microbes.

Adult mouse testicular tissues were fixed in Bouin’s solution overnight, then embedded into the paraffin, and sectioned at 5 μm thickness. For H&E staining, the tissue slides were deparaffinized by xylene, rehydrated with gradient ethanol, and sequentially stained with hematoxylin and eosin. After dehydration and transparency, the tissue sections were sealed with neutral resin. The images were captured via a Nikon ECLIPSE 80i microscope with a DS-Ri1 camera and processed with NIS-Elements BR software.

In vitro tube formation in response to biopsies of SS, ES, DS and AD was studied using 3D fibrin matrices, using a method adapted from Koolwijk et al [25 (link)]. Briefly, fibrin matrices were prepared by addition of thrombin (0.5 U/mL) (MSD, The Netherlands) to a 3 mg/mL fibrinogen (Enzyme Research Laboratories, Leiden, The Netherlands) solution in M199 medium. Hydrogels were pipetted into a 24-well plate (400 μl). After polymerization, thrombin was inactivated by incubating the matrices with HMEC medium. Endothelial cells were seeded at a confluent density of 5.3x10⁴cells/cm² onto the fibrin hydrogels. The endothelial cells in the 24-well-plate were stimulated with HMEC or HMEC supplemented with 5 μg/ml uPAR inhibitor (R&D Systems, Abingdon, UK), 200 μg/ml Avastin® (bevacizumab) (Roche, Welwyn Garden City, United Kingdom) or corresponding isotype control. After 4 h, 0.4um transwells (Cat nr: 3470; Costar Corning Incorporated, Corning, NY) containing 6 mm biopsies of SS, ES, DS and AD were placed above the endothelial cells on the fibrin hydrogels. The sprouts formed by endothelial cells into the fibrin matrices were photographed and analyzed using a Nikon Eclipse 80i microscope and NIS-elements AR software 3.2. The amount of sprouting is expressed as surface area of the sprouts as a percentage of the total surface of the picture.

All phase and fluorescence microscopy was performed on a Nikon Eclipse 80i microscope, and images were captured and processed using NIS-Elements AR 3.0 software. All cultures were fixed with 3.5% formaldehyde. The parasite mitochondrion was detected using the mouse monoclonal antibody 5F4 (α-F₁B ATPase) kindly provided by P. Bradley (unpublished). The parasite Golgi was detected by a rat polyclonal antibody58 (link). HA-tagged proteins were visualized using a rabbit monoclonal antibody (Cell Signaling). Primary antibodies were visualized using either Alexa Fluor 594 or Alexa Fluor 488 secondary antibodies (Life Technologies). Coverslips were washed and mounted on glass slides with 3 μL Vectashield with DAPI (Vector Labs).