Bx 15 microscope

Maternal (heart, SkM) and fetal (placenta) tissue sections were fixed in 10% buffered formalin, dehydrated in graded ethyl alcohol, cleared in xylene, and embedded in paraffin. Paraffin-embedded 5-micron tissue sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome. Slides (at least two per tissue, nine microscopic fields per slide) were imaged at 40x (cardiac and SkM) and 20x (placenta) magnification with an Olympus BX-15 microscope (Center Valley, PA) equipped with a digital camera and Simple PCI software (v.6.0, Compix, Sewickley, PA). Tissue sections stained with H&E were scored for inflammation (i.e., % nuclei) and necrosis (i.e., % tears in tissue). ImageJ version 1.53 (last accessed on Dec 15, 2022, https://imagej.nih.gov/ij/index.html) was used to quantify pixels with nuclei coloration and tissue tears. Briefly, total tissue pixels and nuclei were selected for using color thresholding and percent nuclei quantified [(nuclei pixels ÷ total tissue pixels) x 100]. For the quantification of percent tears [(tears in tissue pixels ÷ total tissue pixels) x 100], total tissue pixels and tears in tissue were selected for using thresholding. Cardiac, SkM, and placental tissue sections stained with Masson’s trichrome were scored for percent fibrotic tissue using ImageJ to quantify fibrotic (blue) pixels [(blue pixels ÷ total tissue pixels) x 100].

T. cruzi trypomastigotes were labeled with 5 µM SYTO^®11 (binds DNA, Molecular Probes-Invitrogen, Eugene, OR) or 5 µM carboxyfluorescein succinimidyl ester (CFSE, binds amines, Invitrogen) for 20 min at 37oC. THP-1- or BM- derived mφs were infected and incubated with labeled T. cruzi trypomastigotes, as above. Cells were washed, and SYTO^®11 or CFSE fluorescence as an indicator of parasite uptake was determined by using an Olympus BX-15 microscope equipped with a digital camera (magnification 40X). Cells infected with CFSE-labeled parasites were also fixed with 2% paraformaldehyde and visualized on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) acquiring 20,000 events. Further analysis was performed by using FlowJo software (ver. 7.6.5, Tree-Star, San Carlo, CA). Mean Fluorescence intensity (MFI) of CSFE positive cells was used as a relative marker of parasites per cell.
Total DNA from normal and infected cells was isolated by using TRIzol reagent (Life Technologies, Grand Island, NY). Total DNA (100 ng) was used as a template in a quantitative PCR (qPCR) on an iCycler thermal cycler with SYBR Green Supermix (Bio-Rad) and oligonucleotide pairs specific for Tc18S ribosomal DNA (Table S1). Data were normalized to murine or human GAPDH, and fold change calculated as 2^−ΔΔCt, where ΔΔC_t represents the C_t (sample) - C_t (control).

Tissue sections were fixed in 10% buffered formalin for 24 h, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Five-micron tissue-sections were stained with hematoxylin and eosin, and evaluated by light microscopy using an Olympus BX-15 microscope equipped with a digital camera. In general, we analyzed each tissue-section for >10-microscopic fields (100× magnification), and examined three different tissue sections/mouse (4 mice/group) to obtain a semi-quantitative score of parasitic pseudocysts (foci). Myocarditis (presence of inflammatory cells) was scored as 0 (absent), 1 (focal or mild with ≤1 foci), 2 (moderate with ≥2 inflammatory foci), 3 (extensive with generalized coalescing of inflammatory foci or disseminated inflammation), and 4 (diffused inflammation with severe tissue necrosis, interstitial edema, and loss of integrity) [38] (link). Inflammatory infiltrates was characterized as diffused or focal depending upon how closely the inflammatory cells were associated [39] (link).