Phosphomolybdic phosphotungstic acid solution

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).

Four weeks after AntCaNtide (50 μg/kg), tat-CN17β (50 μg/kg) or NaCl 0.9%, intra-cardiac injection, the hearts were immersion fixed in 10% buffered paraformaldehyde. The tissues were embedded in paraffin, cut at 5 μm, and processed. For Masson trichrome staining of collagen fibers, slides were stained with Weigert Hematoxylin (Sigma-Aldrich, St. Louis, MO) for 10 min., rinsed in PBS (Invitrogen) and then stained with Biebrich scarlet-acid fuchsine (Sigma-Aldrich St. Louis, MO) for 5 min. Slides were rinsed in PBS and stained with phosphomolybdic/phosphotungstic acid solution (Sigma-Aldrich St. Louis, MO) for 5 min. then stained with light green (Sigma-Aldrich) for 5 min. Slides were rinsed in distilled water, dehydrated with 95% and absolute alcohol and a coverslip was placed. For the analysis of cardiomyocytes size, Masson trichrome staining sections were used [37 (link)]. The areas (μm²) of ~100 cardiac myocytes per heart were measured with the public domain Java image processing program Image by an independent operator blind to the study protocol (DS) [44 (link)].

For histological analysis, WT and galectin-3^−/− mice were sacrificed 60 days after pristane injection. Several oil granulomas were excised, washed in cold saline, fixed in buffered 10 % formalin for 24 h at 4 °C, embedded in paraffin and sectioned into 4-m thick slices. The slices were dewaxed using xylene, hydrated in a graded ethanol, and then washed with distilled water. Lastly, the slices were processed routinely with hematoxylin & eosin (H&E) or with Masson’s Trichrome stain. For Masson’s Trichrome stain, briefly, slides were placed in Bouin solution for 1 h at 56 °C, then stained with Weigert hematoxylin (10 min; Sigma-Aldrich, St. Louis, MO), followed by Biebrich scarlet acid fuchsin (5 min; Sigma-Aldrich), phosphomolybdic/phosphotungstic acid solution (10 min; Sigma-Aldrich), and aniline blue (5 min; Sigma-Aldrich).

For histological analyses, animals were sacrificed after 90 days of infection. Livers were removed, cut into 0.5 mm thick slices, washed in cold saline and fixed in Bouin’s fixative. After 6 h of fixation, specimens were dehydrated in alcohol, and embedded in paraffin. Sections of 5 μm were obtained and stained with Hematoxylin-eosin and Masson’s trichrome staining.
For Masson’s Trichrome stain, briefly, slides were placed in Bouin’s solution for 1 h at 56 °C, then stained with Weigert’s hematoxylin (10 min; Sigma-Aldrich, St. Louis, MO), followed by Biebrich scarlet acid fuchsin (5 min; Sigma-Aldrich), phosphomolybdic/phosphotungstic acid solution (10 min; Sigma-Aldrich), and aniline blue (5 min; Sigma-Aldrich).
For α-smooth muscle actin (α-SMA) immunofluorescence, paraffin-embedded sections were dewaxed and hydrated and washed several times in PBS and further treated with 50nM NH₄Cl (30 min), 0.05 % saponin/0.1 M PBS (30 min) and 0.05 % gelatin/0.05 % saponin/PBS (30 min). Cells were incubated with human monoclonal anti-SMA (Sigma-Aldrich, Inc., St. Louis, MO) diluted 1:400 in 0.05 % gelatin/0,05%saponin/PBS as the primary antibody at 4 °C, overnight. Thereafter, sections were incubated with anti-human Alexa Fluor® 546 (Life Technologies, Inc.) diluted 1:1000. Sections were examined by confocal microscopy Olympus FV-1000.