Masson s trichrome staining kit

GSH assay and TUNEL staining kits were purchased from the Beyotime Institute of Biotechnology (#S0053, #C1090 and #P0012S, Shanghai, China). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were purchased from Nanjing Jiancheng Bioengineering Institute (#C009–2 and #C010–2, Nanjing, Jiangsu, China). Elisa kits for ADMA and 4-hydroxynonenal (4-HNE) were purchased from Bio-Techne Co., Ltd. (#NBP2–66728, Minneapolis, MN, USA) and Donggeboye Biological Technology Co., LTD. (#DG30947M, Beijing, China), respectively. The Masson’s trichrome staining kit was obtained from Solarbio Science & Technology Co. LTD (#G1340, Beijing, China). Antibodies against DDAH1, cytochrome P450 2E1 (CYP2E1), glutathione S-transferase A1 (GSTA1) and β-actin were purchased from Signalway Antibody LLC (#37368, #48247, #22536, #21800, Greenbelt, MD, USA). P65, phospho-65, c-jun N-terminal kinase (JNK) and phospho-JNK antibodies were from Cell Signaling Technology (#8242, #3033, #9252, #9251, Danvers, MA, USA). APAP and dihydroethidium (DHE) were purchased from MedChemExpress LLC (#HY-66005, Monmouth Junction, NJ, USA) and Sigma Chemical Co. (#D7008, St. Louis, MO, USA), respectively. All other compounds and chemicals were of analytical purity.

Mice were anesthetized with 5% isoflurane, and hearts were excised 3-4 weeks after TAC surgery. The death of mice was confirmed by lack of breathing and lack of a heartbeat. Before fixing with 4% paraformaldehyde, the heart was perfused with 10 mL cold PBS. Then, the heart was embedded in OCT compound; 7-μm-thick sections were used for staining. Hematoxylin and eosin staining were performed using a Hematoxylin-eosin Staining Kit (Beijing Solarbio Science & Technology Co., Ltd., Cat. #: G1121) to observe the global changes in heart size. After incubation with hematoxylin solution, sections were washed and differentiated, followed by incubation in eosin solution, dehydrated in a series of ethanol solutions (75%-100%), then cleared in xylene and mounted in neutral gum. Masson's trichrome was used to assess collagen density using a Masson's Trichrome Staining Kit (Beijing Solarbio Science & Technology Co., Ltd. Cat. #: G1346) according to the manufacturer's instructions. To evaluate the cardiomyocyte cross-sectional area, FITC labeled Wheat Germ Agglutinin and Alexa Fluor 647 labeled WGA (Thermo Fisher Scientific, Cat. #: W834 and W32466) were used according to the manufacturer's instructions.

The lungs were fixed in 4% paraformaldehyde, embedded in paraffin, and then sliced (4–5 μm) for H&E and Masson’s trichrome staining. For H&E staining, sections were stained in hematoxylin for 5 min and then stained for 2 min at room temperature in eosin. Collagen in lung tissues was demonstrated by Masson’s trichrome staining kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. The collagen fibers were stained blue. The muscle fibers, cytoplasms, celluloses, keratins, and red cells were stained red. The nucleus was blue-brown. H&E and Masson’s staining were photographed using a light microscope.

All specimens were fixed with 4% PFA overnight at 4°C, embedded with paraffin and 4 μm sections were cut. The elastic fiber and collagen staining were performed by using an elastic staining kit (Sigma-Aldrich, HT25A) and Masson’s Trichrome Staining Kit (Solarbio, G1340, Beijing, China). All procedures were performed according to the manufacturer’s instruction.

The paraffin-embedded tissues were cut into 5-μm sections. The sections were dewaxed and rehydrated. The pathological changes in kidney tissues were determined using a hematoxylin and eosin (HE) staining kit (G1120, Solarbio Science & Technology Co., Ltd., Beijing, China) or a Masson's trichrome staining kit (G1340, Solarbio) following the manufacturer’s protocol. All images were photographed using a camera-equipped light microscope (Nikon Instruments Inc., Tokyo, Japan).

Recombinant human transforming growth factor-β1 (TGF-β1) was purchased from Peprotech (United States). Antibodies against collagen Ⅰ, fibronectin, P-Smad3/Smad3, P-Akt/Akt, P-ERK/ERK, P-JNK/JNK, P-P38/P38, GAPDH, E-cadherin, vimentin, and N-cadherin were purchased from Cell Signaling Technology (United States). The β-tubulin antibody was obtained from Proteintech (China). The α-SMA antibody was obtained from Santa Cruz Biotechnology (China). Goat pAbs against Rb IgG (HRP) and Rb pAbs against Ms IgG were obtained from ImmunoWay (China). TRIzol reagent was acquired from Ambion Life Technology (China). DEPC-treated H₂O and RNase Away H₂O were purchased from Life Technologies. Fastking gDNA Dispelling RT SuperMix was purchased from Tiangen (Beijing, China). UNICON® qPCR SYBR Green Master Mix and liposomal transfection reagents were purchased from Yevsen (Shanghai, China). The Masson’s trichrome staining kit was obtained from Solarbio (China). Recombinant Mouse Platelet-derived Growth Factor-BB were purchased from MedChemExpress (China).