Ab68477

The hearts were excised and postfixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 μm. Following deparaffinization and dehydration, the sections were stained with hematoxylin and eosin (HE) and observed under a light microscope (ML31, Guangzhou, China). One pathologist was blinded to MIRI grade. Histological analysis of infarct size, hemorrhage, and leukocyte infiltration was scored as none, weak, moderate, strong, or very strong (score 0, 1, 2, 3, or 4) [13 (link)].
Immunohistochemistry (IHC) staining was carried out using the following antibodies: activating transcription factor 3 (Atf3, YT0387, 1:250, Immunoway, Suzhou, China) and heme oxygenase 1 (Hmox1, ab68477, 1:200, Abcam, Cambridge, UK). For the IHC score, three fields from each sample were selected at random, and the mean score of the 3 fields was the final score [13 (link)]. An immunohistochemical assessment was performed by two investigators blinded to treatment assignment.
Immunofluorescent (IF) staining was carried out using the Hmox1 antibody (ab68477, 1:200, Abcam, Cambridge, UK). Images were captured on a confocal laser microscope (Nikon C2+, Tokyo, Japan).

The homogenate from the liver tissue was collected, and the lysate was used to lyse the hepatocytes for 30 min on ice. The BCA method was used to determine the protein concentration. The sample was added to 5× loading buffer by volume and heated in a water bath at 100 °C for 5 min. The hepatocytes were sonicated by SJIALAB for 2 min (10% ultrasound intensity) and centrifuged for 10 min to obtain the supernatant. The extracted protein was subjected to polyacrylamide gel electrophoresis (80–120 V), transferred to a PVDF membrane, sealed and incubated overnight at 4 °C with the addition of primary antibody of nNOS (3G6B10, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), eNOS (9D10, Invitrogen), iNOS (PA1036, Invitrogen), NF-κB (PA1186, Invitrogen), IκB-α (397700, Invitrogen), Mn-SOD (PA530604, Invitrogen), Cu/Zn-SOD (PA5270240, Invitrogen), CAT (PA259183, Invitrogen), HO-1 (ab68477, Abcam, Cambridge, MA, USA), Nrf2 (ab92946, Abcam), γ-GCS (sc-390811, Santa Cruz Biotechnology, Dallas, TX, USA), NQO1 (ab28947, Abcam) and β-action (MA5157739, Invitrogen). The protein was incubated overnight at 37 °C with the addition of a second antibody (A21241) for 2 h, and color detection and chemiluminescence imaging analysis system (Tanon 6200, ShanghaiTanon Technology Co., Ltd., Shanghai, China) were used to collect images [56 (link)].

The protein secretion of PI3 kinase p85 (PI3K), phospho-PI3K (p-PI3K), heme oxygenase 1 (HO-1) and indoleamine 2,3-dioxygenase (IDO) in cultured mesenchymal stem cells according to the experimental procedures were detected by western blot assay.
Adherent cells were scraped from the culture dishes, and total protein was extracted from the cell pellet with RIPA lysis buffer (89,900, Thermo Scientific). The proteins were separated through a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (ISEQ00010, Merck Millipore). The membrane was blocked and probed with primary antibodies overnight, including PI3 kinase p85 (PI3K, ab133595, abcam), phospho-PI3K (p-PI3K, ab182651, abcam), heme oxygenase 1 (HO-1, ab68477, abcam), indoleamine 2,3-dioxygenase (IDO, ab277522, abcam) and β-Actin (ab8226, abcam) at 1:1,000 dilutions. The membrane was then incubated with the secondary antibodies (115-035-003, Jackson). The signals were scanned for the densitometry and the final blots were quantified with ImageJ Version 2.1.0/1.53c software.