Biebrich scarlet acid fuchsin solution

The mouse lung sections were placed in Bouin’s solution at 56 °C for 1 h and then stained successively with Mayer’s hematoxylin solution for 5 min, Biebrich scarlet-acid fuchsin solution for 10 min, phosphomolybdic acid-phosphotungstic acid for 15 min, and aniline blue for 2 h (staining reagents from Sigma-Aldrich). The sections were examined under a microscope.

We conducted Masson’s trichrome staining in order to examine changes in collagen fibers of dorsal skin as we described previously [2 (link),40 (link)]. Briefly, the sections were immersed with Biebrich scarlet-acid fuchsin solution (Sigma-Aldrich, St. Louis, MO, USA) and reacted with aniline blue (Sigma-Aldrich, St. Louis, MO, USA). Afterwards, the sections were dehydrated and mounted by cover glass using the same mounting medium.

Hearts were embedded in paraffin and 5 μm thick serial sections of the aortic sinus were collected on glass slides [40 (link)]. Sections were deparaffinised and then stained with Weigert’s Hematoxylin (Sigma-Aldrich, St. Louis, MI, USA). The slides were further stained with Biebrich scarlet-acid fuchsin solution and alanine blue (Sigma-Aldrich, St. Louis, MI, USA). Coverslips were attached using DPX mounting media and images were captured using the Olympus DP71 digital camera (Olympus imaging, Centre Valley, PA, USA) mounted on a Leitz Laborlux S bright-field microscope (Leica Microsystems, Concord, ON, USA). Atherosclerotic plaque areas were determined from 12 serial sections per mouse. The atherosclerotic volumes were determined using ImageJ software (Version1.15j8, https://imagej.nih.gov/ij/index.html (accessed on 8 November 2022)), as previously described [40 (link)].

0 = none to minimal—No inflammation within the muscle bundles or inter-bundle connective tissue; occasional mononuclear inflammatory cells may be present but no obvious aggregations.
1 = mild—Occasional mononuclear inflammatory cells in the inter-bundle connective tissue with focal aggregations of mononuclear inflammatory cells.
2 = moderate—Multiple foci of mononuclear inflammatory cell infiltration in the inter-bundle connective tissue; occasional mononuclear inflammatory cells between individual muscle fibres.
3 = severe—Multiple large foci of mononuclear inflammatory cell infiltration in the inter-bundle connective tissue extending into the intra-bundle connective tissue with expansion of the inter-bundle and intra-bundle spaces.
For Masson’s trichrome staining, frozen muscle (diaphragm, 7 mice/group) sections were stained in Weigert’s iron haematoxylin working solution (Sigma-Aldrich), Biebrich Scarlet-Acid fuchsin solution (Sigma-Aldrich), Phosphotungstic/phosphomolybdic acid solution, aniline blue solution, and 1% acetic acid solution (Sigma-Aldrich).
For quantitative analysis of the fibrotic area, bright field images of Masson’s trichrome-stained sections were captured using a digital camera (DFC295; Leica, Germany) at 200- fold magnification and the positive areas in five fields/section (TBD) were measured using ImageJ software (National Institute of Health, USA).

Microscopy slides with tissue sections were removed from the freezer and air-dried at RT and then immersed for 5 min in distilled water. Sections were immersed in preheated Bouin’s solution (Sigma #HT10132) for 15 min at 56°C and then washed under tap water at RT. Sections were then immersed in haematoxylin solution (Sigma Aldrich, #51275) for 5 min, washed for 5 min under running tap water and rinsed with distilled water. Sections were then stained with Biebrich scarlet-acid fuchsin solution (Sigma Aldrich, #HT15) for 5 min and rinsed with distilled water. Microscopy slides were immersed into phosphotungstic/phosphomolybdic acid working solution (Sigma Aldrich, #HT15) for 5 min and then moved into aniline blue solution (Sigma Aldrich, #HT15) for another 5 min, followed by acetic acid (1%) treatment for 2 min, and were then rinsed with distilled water. After the staining, sections were treated with an incremental gradient of ethanol (1 min each, 70%, 2× 95%, 2× 100%), followed by 5 min immersion in xylene. Microscopy slides were finally mounted with Entellan (Merck #1079600500).

The slides were prepared as described in the Additional file 1 and stained in 1% Biebrich scarlet-acid fuchsin solution for 10 min (Sigma, USA). Tissue was differentiated in the phosphomolybdic–phosphotungstic acid solution for 10 min, and the slide transferred into Light green 2% solution, followed by a wash in distilled water.