Vs200 fluorescent slide scanner

Slides from animals survived 1 week postinjection were used to quantify macrophage density. Two locations from the apex, mid ventricular region, and base were taken, and 2 slides were taken at each location with a total of 4 to 6 tissue sections for each stain. Immunohistochemistry was performed by using antibodies for CD68 (1:300; Abcam) and CD163 (1:300; Bio-Rad) incubated with goat anti-rabbit immunoglobulin G conjugated Alexa Fluor 750 (1:500; Invitrogen) and goat anti-mouse immunoglobulin G conjugated Alexa Fluor 594 (1:500; Invitrogen), respectively. Slides were then mounted using Fluoromount (Sigma-Aldrich) and scanned at 20× magnification using an VS200 fluorescent slide scanner (Olympus Life Science). Macrophages were quantified as total number of CD68⁺ cells colocalized with 4′,6-diamidino-2-phenylindole, total number of CD163⁺ cells co-localized with 4′,6-diamidino-2-phenylindole, and ratio of total number of CD163/CD68 using cell counter analysis in the QuPath version 0.4.3 software.

Fluorescein labeled Griffonia Simplicifolia Lectin I isolectin B4 (1:75; Vector Laboratories) was used to identify endothelial cells within the right ventricle, α-smooth muscle actin (α-SMA) (1:75; Dako) was used to identify smooth cells, and Hoechst 33342 (1:1000; Life Technologies) was used to identify nuclei. Slides were then mounted by using Fluoromount and scanned at 20× magnification using an Olympus VS200 fluorescent slide scanner. Arteriole and myofibroblast density was quantified by manually counting in 5 evenly distributed regions throughout the right ventricle in 6 slides (evenly spaced throughout the RV mid ventricular region) using Fiji. Arterioles were identified based on the following criteria: 1) co-staining of isolectin and α-SMA; 2) presence of a visible lumen; and 3) Feret diameter >10 μm as previously described.¹⁰ (link)^,14 (link) Capillaries were identified as percent positive area of isolectin not co-localized with α-SMA. Myofibroblasts were identified as cells staining positive for α-SMA and not found in vessels.¹⁵