Ab222941

Atria were collected, fixed, and sectioned for histological analysis of alpha smooth muscle actin (αSMA; A5228, Sigma), inflammatory infiltrates (hematoxylin and eosin (H&E), Sigma), collagen (ab210579, Abcam; LS-F5563-1, LifeSpan BioSciences; picrosirius red, Thermo Fisher Scientific), wheat germ agglutinin (Thermo Fisher Scientific), connexin 40 (ab1726, Millipore Sigma), and connexin 43 (C6219, Sigma). Images were quantified using ImageJ 37 (link), 38 (link). Left atria collagen content was confirmed using an assay for hydroxyproline (ab222941, Abcam), an indicator of collagen content/turnover 39 (link), 40 (link).
Homogenized atrial tissue was analyzed for markers associated with RTFI00051 using enzyme-linked immunosorbent assays (ELISAs). These assays included matrix metalloproteinase 2 (MMP2; RTFI00042, AssayGenie), MMP9 (RTFI00080, AssayGenie), tissue inhibitor of metalloproteinases 2 (TIMP2; RTFI00051, AssayGenie), TIMP3 (RTFI01169, AssayGenie), and αSMA (MBS2703516, MyBioSource).

A hydroxyproline assay was performed to confirm the total tendon collagen content. The sacrificed tissue was rapidly frozen in liquid nitrogen and stored at −80 °C. Tissue disruption and experimental methods were performed following the manufacturer’s instructions for a hydroxyproline colorimetric assay (Abcam; ab222941). After breaking the tissue, proteins were treated with 10 N NaOH and HCl and then centrifuged at 10,000× g for 5 min. After collecting the supernatant, an oxidation mixture was added and reacted at room temperature for 20 min. The developer and DMAB concentrate were added at 50 μL per vial and reacted at 65 °C for 45 min. Measurements were obtained at 560 nm using a Synergy Mix Multi-Mode Reader (BioTek).

Frozen mouse kidney tissue was made into homogenate (0.1 M Tris/HCl, pH 7.4, containing 0.5% Triton X-100, 5 mM β-ME, and 0.1 mg/ml PMSF) and centrifuged at 1,400 × g at 4℃ for 5 min to collect the supernatant. The contents of superoxide dismutase (SOD, #K335, BioVision, Milpitas, CA, USA), malondialdehyde (MDA, #K739, BioVision), interleukin (IL)-6 (ab100713, Abcam Inc., Cambridge, MA, USA), monocyte chemotactant protein-1 (MCP-1, ab100722, Abcam), and hydroxyproline (ab222941, Abcam) were determined adhering to the instructions of the ELISA kits.

For measurement of hydroxyproline in lung tissue, it was performed using hydroxyproline colorimetric assay kit (ab222941, Abcam). For sample preparation, the lung tissues were reacted with 10 N NaOH for 1 h. Additionally, then, it was neutralized with 10 N HCl. Afterward, we followed the manufacturer’s protocol. Briefly, oxidation mixture was added to supernatant and reacted for 20 min. The developer and DAMB were added per well, and reacted at 65 °C for 45 min. Finally, it was measured at 560 nm by a microplate reader (BioTek, Winooski, VT, USA).

Mice were weighed for body weight changes. After the mice were euthanized, the colon was removed entirely, and the length and weight were measured. The length from cecum to the anus was used to determine the length of the colon. The complete mouse colon was excised for historical analysis, and segments of the transverse tissues (1 cm) were then fixed in 10% buffered formalin, paraffin-embedded, and stained with hematoxylin and eosin. Zeiss Axio Scan.Z1 slide scanner was used to collect and digitize all histological sections, and ZEN 3.1 (blue edition) software was used for analysis. The thickness of the mucosa and muscularis propria of each mouse was calculated as the mean value of five distinct points. Sirius red staining (ab150681, abcam) was used to evaluate fibrosis. A hydroxyproline assay kit (ab222941, abcam) was used to obtain the collagen in the tissue according to the protocol of the manufacturer. A Zeiss Axio Scan.Z1 slide scanner was used to read the stained sections. Using ZEN 3.1 (blue edition) software, the quantified outcomes of Sirius red staining were presented as the mean density of five randomly selected fields from each group.

The leukogram was performed using blood smears, stained (Panotico Fast, Laborclin, Brazil) and viewed under an optic microscope (Nikon, Japan) for differential count of neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine, urea, creatine kinase (CK) creatine kinase MB (CKMB), and C-reactive protein were evaluated using the respective kits (K048-6, K049-6, K067-1, K056-1, K010, K069 and K059, Bioclin, MG, Brazil, respectively) and cardiac troponin I was determined using kit (Elabscience Biotechnology Co. Ltd., Wuhan, China). All biochemical concentrations were determined according to the instructions for each kit used. The concentration of hydroxyproline in the rat cardiac tissues were measured with an ELISA kit (ab222941 Abcam, Cambridge, UK) following the manufacturer’s protocol.

The hydroxyproline content was assessed as an index for collagen and fibrosis content in cardiac cell lysates using a colorimetric hydroxyproline assay kit (Abcam, USA, Ab222941). The assay was performed according to the manufacturer’s instructions.