Picrosirius red staining kit

5 µm lung and heart sections were cut, from paraffin blocks and mounted on Superfrost® microscope slides (Sigma-Aldrich, Irvine, UK).
Vascular thickening was determined by smooth muscle actin antibody (ab5694, Abcam, Cambridge, UK) staining, thickening was characterised by an increase in the vessel wall diameter of more than 50% of the arterial wall or complete occlusion. The number of remodelled vessels over the total number of vessels present in a lung section was determined. Sections were analysed in a blinded manner.
Heart sections were stained using a common regressive Haemotoxylin and Eosin (H&E) staining methodology to determine gross morphology and cardiomyocyte cross-sectional area. High-power images of five areas randomly distributed across ventricles were analysed by ImageJ software. Mean cardiomyocyte size was determined by measuring the cross sectional area of minimally 20 transversally cut cardiomyocytes at the level of the nucleus.
Picro-sirius red staining was used for analysis of cardiac fibrosis using a Picro-sirius red staining kit (Abcam, Cambridge, UK) as per manufacturer's instructions. High-power images were acquired of five random areas per ventricle. Using ImageJ IHC tool, the percentage tissue area positive for collagen (pink) was determined.

A liver specimen (approximately 5 mm × 5 mm) taken from the right lobe near the portal vein was fixed in 4% (w/v) paraformaldehyde, dehydrated, and embedded in paraffin as previously described.⁷⁵ The paraffin-embedded liver sections (5 μm) were deparaffinized, rehydrated, and stained with H&E, Sirius Red (Picro Sirius Red Staining Kit, ab150681; Abcam, Cambridge, UK), or Trichrome Stain (Masson’s) (SDHT15; Sigma-Aldrich, Burlington, MA) according to the manufacturer’s instructions. For Oil Red O staining, liver embedded in optimal cutting temperature compound (OCT compound) was immediately frozen on dry ice. Frozen sections (7 μm) were obtained using a Leica CM 1900 Cryostat (Leica Biosystems, Nussloch, Germany). Rehydrated sections were rinsed with 60% isopropanol and stained for 20 minutes with 0.3% Oil Red O solution, followed by rinsing 3 times in 60% isopropanol and twice in double-distilled water. Slides were further counterstained with hematoxylin and mounted.