Sirius red

Myocardial fragments were fixed in 4% buffered formalin, processed by a standard technique, and paraffin-embedded sections (3–4 μm thick) were stained with hematoxylin and eosin, Masson’s trichrome (Bio-Optica), Sirius Red (Bio-Optica) and used for histological examination. Masson’s trichrome-stained sections were used in total for morphometric evaluation of percent fibrosis at × 100 using the Image-Pro Plus program (version 6.0.0.260; Media Cybernetics, USA). Sirius Red-stained sections were used for a semi-quantitative 5-point assessment of the amyloid deposits distribution in the interstitium around blood vessels and cardiomyocytes: 0—no deposits; 1—deposits were around single cardiomyocytes; 2—around small groups of cardiomyocytes; 3—around half of cardiomyocytes and single vessels; 4—around more than 75% of cardiomyocytes and blood vessels. The slides were examined by light microscope Leica DMRB with Leica DFC495 camera and Leica PL FLUOTAR 10 × /0.3 and Leica N PLAN 40 × /0.65 (Leica Microsystems Gmbh, Austria).

To further evaluate the degree of fibrosis collagen accumulation in the lung tissues, sections were stained with Masson trichrome and Sirius red according to the manufacturer’s protocols (Bio-Optica, Milan, Italy), as previously described [31 (link)]. Images were shown at 20× magnification (50 μM of the Bar scale) using a Nikon Eclipse Ci-L microscope.