Dab reagent

Transformed E104 E. coli were propagated in Terrific Broth media (1% inoculum) to an O.D.₆₀₀ of 2. At this time, the culture was induced with 0.1% l-arabinose (+) (Sigma) and incubated at 25 °C overnight. After centrifugation, supernatants were analyzed by Western blot to confirm the presence and size of the fusion proteins. Briefly, induced supernatants were loaded on a SDS-PAGE gel and transferred to a nitrocellulose membrane. Diabody and diabody fusion proteins were detected with an anti-Histidine (His) antibody (GE Healthcare, Buckinghamshire, UK) at a 1/1000 dilution followed by a sheep anti-mouse IgG coupled to horseradish peroxidase (HRP) (Sigma) at a 1/1000 dilution. The signal was developed using the DAB reagent (Sigma).

Continentalic acid (purity 99.9%) was received from Prof. Yeong Shik Kim, Emiritus Professor, College of Pharmacy, Seoul National University, Korea. All the chemicals and reagent included in this study such as dexamethasone and LPS were obtained from the Sigma Aldrich (St. Louis, MO, USA). The cytokines were analyzed in lung tissue using ELISA Kits obtained from the eBioscience (eBioscience, Inc. USA). The primary and secondary antibodies for Nrf2 and iNOS were obtained from the Santa Cruz (Santa Cruz Biotechnology, Inc). The DAB reagent was obtained from the Sigma Aldrich (St. Louis, MO, USA). The NGS (normal goat serum) and AB complex used in the immunohistochemistry were obtained from (SCBT, U.S.A).

Total protein from isolated ovaries was precipitated after RNA and DNA isolation using TriPure total RNA isolation kit according to the manufacturer’s procedure (Roche Molecular System, USA) and its concentration was measured using Bradford method as described previously [23 (link)]. Twenty five μl of each protein sample (1 μg/μl) were mixed with 25 μl Laemmli sample buffer supplemented with 2-mercaptoethanol at a final concentration of 7.5% (vol/vol). The samples were heated for 15 min at 65°C, separated by 10% SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene, NH). The filters were blocked by incubation for 1 h in PBS with 5% nonfat milk. Blots were then washed in PBS-Tween and immunoblotted with primary antibody against mouse chemerin (Abcam, Cambridge, UK, Art No: ab112450) at 1:500 ratio. Detection of primary antibody was done using goat anti rabbit HRP-conjugated antibody (Abcam, Cambridge, UK, Art No; ab98467) at 1:1000 ratio and DAB reagent (Sigma Aldrich, Germany). Densitometric quantification of chemerin proteins in relation to GAPDH as calibrator was performed using Image J software (National Institutes of Health). Western blot was done in three independent experiments for each sample.

Mice liver tissues were fixed in 10% buffered formalin and embedded in paraffin. Liver sections were routinely stained Hematoxylin and Eosin and Sirius Red. Frozen sections from the different groups of mice were stained for Oil Red O. Diagnosis of NASH was based on the presence of steatosis, inflammation and hepatocyte ballooning. The severity was assessed by using the NAS scoring system [13] (link).
For Immunohistochemistry, liver sections were incubated with primary monoclonal antibodies against Alpha-Smooth Muscle Actin (αSMA) (DakoCytomation, Carpinteria, CA), p-AKT Ser473, β-Catenin (Cell Signaling, Boston, MA), p-c-Myc Ser373 (Biorbyt, Cambridge, UK) and Glypican-3 (Cell Marque, Rocklin, CA). Antibodies against Ki67 and F4/80 were from ABCAM, Cambridge, UK. In Situ Cell Death (TUNEL) kit was from Roche, Manneheim, Germany. Detection of positive staining was performed by using DAB reagent (Sigma-Aldrich, St. Louis, MO).
Quantitative morphometry was performed as previously described [14] , [15] (link).

Hydrogen peroxide (H₂O₂) was detected with 3,3′-diaminobenzidine tetrachloride (DAB) reagent (Sigma-Aldrich, Darmstadt, Germany) [61 (link)]. Roots were immersed in 0.05% DAB (pH 3.8) for 3 h at room temperature in the dark. Subsequently, roots were washed in distilled water and photographed.

Fresh bone tissue was fixed in 10% neutral buffered formalin, decalcified by formic acid, and embedded in paraffin. Thin sections (5 µm) were mounted onto tape adhesive slides. Following deparaffinization and rehydration with ethanol, endogenous peroxidase was blocked using 3% hydrogen peroxide/distilled water. Antigen retrieval was performed using a citrate buffer (BioGenex) for 10 s at 100 °C, then slides were cooled and rinsed in distilled water and Tris-buffered saline/Tween-20 (TBS-T). After blocking in 5% normal goat serum (Vector Labs), sections were incubated with anti-phospho-p44/42 MAPK ERK1/2 rabbit monoclonal antibody (Thr202/Tyr204; diluted 1:200, Cell Signaling Technology, USA #4376) overnight at 4 °C or monoclonal rabbit IgG isotype control. Subsequently, biotinylated goat anti-rabbit IgG (1:100, Vector Labs) was applied for 30 min, before slides were incubated with ABC Elite Standard (Vector Labs) for 30 min. p-ERK1/2 was visualized by a 5-min incubation in DAB Reagent (Sigma) and subsequent hematoxylin counterstain. Slides were dehydrated, cleared in xylene and mounted with coverslips with Permount. Paraffin embedded colon cancer cells (SW48, carrying a p.Q56P mutation in MAP2K1) was used as positive control^{13 (link)}. Normal bone from patients undergoing surgical resection for unrelated indications served as negative control (Fig. 4a).