Masson s trichrome stain kit

Tissue sections were stained with hematoxylin & eosin (H&E) (hematoxylin, GHS132-1L; eosin Y solution, HT110132-1L; Sigma-Aldrich, St. Louis, MO) for routine light microscopy evaluation. The Masson’s trichrome stain kit (ab150686, Abcam, Cambridge, UK) to detect cardiac fibrosis was used according to the manufacturer’s instructions. Picro Sirius red staining (Direct red 80, 65548, Sigma-Aldrich, St. Louis, MO) and Picric acid (10015714, Xilong, China) was also applied to evaluate the fibrotic area, appearing as red color. Histopathological scores were graded as 0 “-” (no apparent change), 1 (minimal), 2 (moderate), 3 (marked), and 4 (severe) based on the increasing extent and/or complexity of morphological changes^{124 (link),125 (link)}. More details for grading standards are described in Supplementary Table 2.
The stained tissue sections were captured under an Olympus BX53 digital microscope with the cellSens digital imaging software (Olympus, Tokyo, Japan), scanned on a Leica Aperio AT2 scanner (200x magnification; Leica Biosystems), and analyzed with the Halo software v3.0.311.217 (Indica Labs) with Area Quantification (v2.1.3.0; Indica Labs) and CytoNuclear (v2.0.5.0; Indica Labs) algorithm.

Glutathione (reduced form) was purchased from Wako Pure Chemical (Osaka, Japan). Pectin and agar were purchased from Yakuri Co., Ltd. (Osaka, Japan). Sodium alginate, PEG (average molecular weight: 8000), sodium nitrite, crystal violet, Lugol’s solution, 2,2,2 tribromoethanol, tert-amyl alcohol, Mayer’s hematoxylin, and eosin-Y disodium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bacto™ tryptic soy broth (TSB) and Difco™ cetrimide-agar media were purchased from BD Biosciences (San Jose, CA, USA). A Twort’s Gram stain set was purchased from Newcomer Supply (Middleton, WI, USA). Masson’s trichrome stain kit was purchased from Abcam (Cambridge, MA, USA). The LIVE/DEAD^®BacLight™ bacterial viability kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other reagents and solvents were of the highest analytical grade commercially available.