Ndp viewer

Tissue volumes were determined from serial sections using the Cavalieri method48 . In brief, cross-sectional areas on serial sections were determined and volumes calculated using the spacing between sections. Arterial remodelling was assessed in the central 2/4 of the decidua basalis on sagittal sections to avoid inclusion of veins that have been shown to be more peripherally located39 (link). Three to five implantation sites (corresponding to one uterine horn chosen at random) were analysed per litter. For each implantation site, the five largest vessels were measured in triplicate (three sections 49 μm apart from each other). For all stereological measurements, uterine arteries were tied before dissection to minimize bleeding. Size of lumens and vessels was determined by measuring cross-sectional areas with NDP viewer (Hamamatsu) as this was found to yield more reproducible results than measuring diameters. Differences in size and relative thickness of vessels were confirmed by smooth muscle actin staining. For determination of trophoblast invasion, H&E and cytokeratin stainings were used and analysed as described before8 (link) using the most distal section from the fetus that contained fetal red blood cells as a marker for the base of the ectoplacental cone and the most distal section positive for cytokeratin staining as its apex.

Ears from 8- to 10-week-old mice were collected and fixed in 4% paraformaldehyde overnight at 4°C. For each ear, the dorsal skin was separated from the rest of the tissue (central cartilage and the adhering ventral skin). Whole dorsal ear skins were fixed in methanol for 1 hour at –20°C and then blocked with 3% milk and 0.2% Triton X-100 for 1 hour at room temperature. They were then incubated with a polyclonal goat anti-LYVE1 antibody (1:200; AF2125, R&D Systems) and rat anti-CD31 antibody (1:150, DIA-310, Dianova) overnight at room temperature. After washing in PBS, an Alexa Fluor 488–coupled rabbit anti-goat antibody (1:200; A21222, Invitrogen) was added for 2 hours at room temperature. After washes with PBS, Alexa Fluor 546–coupled goat anti rat antibody (1:200; A11081, Invitrogen) was incubated at room temperature for 2 hours. Slides were then washed with PBS and mounted using Dako fluorescent mounting medium (Dako). Slides were scanned using a Nanozoomer 2.0 scanner (Hamamatsu) and visualized using NDPviewer (Hamamatsu). The lymphatic vessel frequency, density, and thickness were quantified using ImageJ software.

Podocyte density was calculated in each glomerulus as p57 + cell number/glomerular area. Glomerular planar area and glomerular diameter were calculated using a virtual slide system (NDP viewer, Hamamatsu Photonics). We measured all the glomeruli encountered in each section (on average, 93.1 glomeruli/animal). CD44-positive glomeruli were quantified by the incidence of glomeruli having CD44 + cells. Similar to p57, we analyzed CD44 + glomeruli among all the glomeruli encountered in each section (on average, 97.7 glomeruli/animal). To express the degree of glomerular injury in each animal, glomerular scoring was performed. Glomerular lesions were divided into four grades using PAM-stained slides depending on the glomerular area involved by crescent; S0 (no change), S1 (< 50%), S2 (> 50%), S3 (global sclerosis). All the glomeruli in the single section in each animal were individually scored, and the average value in each animal was calculated. On average, over 90 glomeruli per section were examined. Fibrinoid exudate was estimated by MT staining showing red-colored amorphous deposition in the tuft or urinary space. The incidence of fibrin-positive glomeruli per section was calculated.