Zen 2 blue edition version 2

We analyzed 3–5 outflow veins per animal per group for histomorphometric analysis. Sections were stained for Hematoxylin and Eosin (H and E) to assess venous remodeling. Images were digitized to capture a minimum of 1936 × 1460 pixels covering one entire cross-section utilizing an M2 Microscope (Carl Zeiss) with an Axiocam 503 color camera (Carl Zeiss). These slides were analyzed using ZEN 2 blue edition version 2.0 (Carl Zeiss) as described elsewhere^{4 (link),11 (link),16 (link),30 (link)}. We measured the lumen vessel area, neointima, media, and adventitia areas, along with cell density in each of the layers.

Hematoxylin and eosin stained outflow veins to assess venous remodeling were assessed. Images were digitized to capture a minimum of 1936×1460 pixels, covering one entire cross‐section using an M2 Microscope (Carl Zeiss) with an Axiocam 503 color camera (Carl Zeiss). These slides were analyzed using ZEN 2 blue edition version 2.0 (Carl Zeiss), as described elsewhere.⁹, ¹⁰, ¹⁴, ¹⁵, ¹⁶ In addition, slides stained for Masson trichrome, TUNEL, smooth muscle actin (SMA), cluster of differentiation (CD) 68, inducible NO synthase (iNOS), myosin heavy chain (MYH11), arginase 1 (Arg‐1), fibroblast specific protein‐1 (FSP‐1), phospho Mothers Against Decapentaplegic Homolog 3 (pSMAD3), MMP‐9, collagen 1, and Ki‐67 were digitized and quantified, as described elsewhere.⁹, ¹⁰, ¹⁴, ¹⁵, ¹⁶ The index for each stain (TUNEL, SMA, CD68, iNOS, MYH11, Arg‐1, FSP‐1, pSMAD3, MMP‐9, collagen 1, and Ki‐67) was calculated as follows: (number of total cells positive for that stain/total number of cells)×100.

Sections that were stained with immunobiological stains and immunofluorescent stains were digitized to capture a minimum of 1936 × 1460 pixels covering one entire cross-section utilizing an M2 Microscope (Carl Zeiss) with an Axiocam 503 color camera (Carl Zeiss). These slides were analyzed using ZEN 2 blue edition version 2.0 (Carl Zeiss) as described elsewhere^{4 (link),11 (link),16 (link),30 (link)}. Staining index was obtained by number of positive cells/ total number of cells × 100.