Weigert hematoxylin

At the fourth week after MI modeling, the myocardial tissues of mice in each group were fixed for 16–18 h using 4% paraformaldehyde solution, hydrated, embedded into paraffin, sectioned at a thickness of 10 μm, and washed with running water and distilled water for 3 min respectively. Then, the cells were stained using Weigert hematoxylin for 5 min (1.15973, Sigma, St Louis, MO, USA), stained with Masson ponceau acid fuchsin solution for 5 min, soaked with 2% glacial acetic acid for 30 s, differentiated with phosphomolybdic acid solution, and stained with aniline blue or light green solution for 5 min. Subsequently, the tissue sections were soaked in 2% glacial acetic acid for 15 s, dehydrated, cleared and fixed with neutral balsam, followed by cover-slipping and observation under an optical microscope (DSX100, Olympus, Tokyo, Japan). The collagen-rich deformed infarct area was stained blue and the cell matrix was stained red. Five visual fields were randomly selected from each section and then analyzed using the ImageJ software. The degree of myocardial fibrosis was expressed as the percentage of fibrotic area in the total area.

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).