oil red o staining kit oro  (Beyotime)

Journal: Hepatology (Baltimore, Md.)

Article Title: MIF-mediated crosstalk between THRSP + hepatocytes and CD74 + lipid-associated macrophages in hepatic periportal zone drives MASH

doi: 10.1097/HEP.0000000000001429

Figure Lengend Snippet: AAV8- Thrsp -OE aggravated MASH via PA accumulation and MIF-mediated CD74 + LAMs recruitment in the PP zone. (A, B) Thrsp OE was confirmed by WB (A) and qPCR (B) (n=3/group, mice). (C) Representative livers, (D) liver weight to body weight ratios (n=7/group, mice), (E) insulin levels and HOMA-IR (n=5–6/group, mice), (F) representative H&E, ORO and sirius red staining of the liver sections, (G) liver TG and TC contents (n=7–8/group, mice), (H) serum ALT and AST levels (n=7–8/group, mice), (I–K) relative mRNA levels of lipogenetic and uptake genes (I), proinflammatory genes (J), and profibrotic gene (K) in livers of NC and Thrsp -OE mice (n=3/group, mice). (L) The volcano map of hepatic FFA content in the NC and Thrsp -OE mice (n=3/group, mice). (M) The content of palmitic acid in the NC and Thrsp -OE mice (n=3–4/group, mice). (N) The serum MIF levels in the normal CD and custom WD fed NC and Thrsp -OE mice. (O) Our results of snRNA-seq showed expression levels of LAMs and Cd74 + LAMs in the livers of NC and Thrsp -OE mice (n=3/group, mice). (P) Representative mIHC images and responding quantification in the NC and Thrsp -OE mice (more than 6 fields of 3 replicates for each group). Scale bar, 50 μm. (Q, R) WB was used to confirm Thrsp overexpression (Q) or knockdown (R) in the AML12 cells treated with/without PA. (S, T) The MIF concentration in the culture supernatant of AML12 cells and RAW264.7 cells, which were treated with culture supernatant from anterior AML12 cells with/without PA treatment. N=3/group. Data were presented as the mean±SEM. p Values were calculated by unpaired t test (2 groups), 1-way ANOVA followed by Tukey multiple comparison test (≥3 groups). * p <0.05, ** p <0.01, and *** p <0.001. SDS, PP marker gene. PA, palmitic acid (PA, C16:0), 0.5 mM. Abbreviations: CD, chowdiet; FFA, free fatty acids; LAMs, lipid-associated macrophages; MASH, metabolic dysfunction–associated steatohepatitis; MIF, macrophage migration inhibitory factor; mIHC, multiplex immunohistochemistry; NC, negative control; ORO, oil red O; PP, periportal; qPCR, quantitative PCR; TC, total cholesterol; TG, triglycerides; Thrsp-OE, Thrsp-mediated overexpression; WB, western blot; WD, western diet.

Article Snippet: Subsequently, they were stained using the ORO stain kit by the manufacturer’s protocols (Sangon Biotech)., The primers for Thrsp overexpression plasmid construction and siRNA target sequences for Thrsp knockdown were listed in Supplemental Tables S4, http://links.lww.com/HEP/J840 , and S6, http://links.lww.com/HEP/J840 .

Techniques: Staining, Expressing, Over Expression, Knockdown, Concentration Assay, Comparison, Marker, Migration, Multiplex Assay, Immunohistochemistry, Negative Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: MIF-mediated crosstalk between THRSP + hepatocytes and CD74 + lipid-associated macrophages in hepatic periportal zone drives MASH

doi: 10.1097/HEP.0000000000001429

Figure Lengend Snippet: THRSP increased hepatic lipogenesis by activating AKT/mTOR signaling pathway and enhancing FASN protein stability. (A, B) WB was used to confirm the Thrsp overexpression and knockdown by siRNA. Representative Oil Red O staining in the primary hepatocytes of the indicated groups (more than 6 fields of 3 replicates for each group). Scale bars, 100 μm. (C, D) The expressions of the indicated proteins in the Thrsp -transfected AML12 cells treated with (A) or without (B) HGHI. (E, F) The quantification of A and B (3 independent experiments conducted). (G) The expressions of THRSP and FASN co-expressed in the livers of the indicated groups. Scale bar, 20 μm. (H) The quantification of G and the colocalization efficiency (Pearson R , overlap ratio) between THRSP and FASN (more than 6 fields of 3 biological replicates for each group). (I) Co-IP assay revealing the interaction of THRSP and FASN. (J, K) FASN expression and quantification in the indicated groups. Data were presented as the mean±SEM. All statistical analyses were carried out by 1-way ANOVA followed by Tukey multiple comparison test (E, F), 2-way ANOVA followed by Sidak multiple comparison test (K), and unpaired t test (H). ** p <0.01, and *** p <0.001. Abbreviations: AKTi mix, AKT inhibitor mixtures; CHX, cycloheximide (10 μM). Co-IP, co-immunoprecipitation; FASN, fatty acid synthase; HGHI, high glucose (30 mM) and high insulin (100 nM).

Article Snippet: Subsequently, they were stained using the ORO stain kit by the manufacturer’s protocols (Sangon Biotech)., The primers for Thrsp overexpression plasmid construction and siRNA target sequences for Thrsp knockdown were listed in Supplemental Tables S4, http://links.lww.com/HEP/J840 , and S6, http://links.lww.com/HEP/J840 .

Techniques: Over Expression, Knockdown, Staining, Transfection, Co-Immunoprecipitation Assay, Expressing, Comparison, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: MIF-mediated crosstalk between THRSP + hepatocytes and CD74 + lipid-associated macrophages in hepatic periportal zone drives MASH

doi: 10.1097/HEP.0000000000001429

Figure Lengend Snippet: Hepatocyte-specific Thrsp -HKO mice ameliorated MASH by reducing hepatic PA content and CD74 + LAMs recruitment mediated by MIF in the PP zone. (A) Representative livers, (B) liver-to-body weight ratio, (C) THRSP protein expression level, (D) the quantification of C, (E) representative H&E, Oil Red O (ORO), and sirius red staining of the liver sections, (F) serum ALT (left) and AST (right), (G) liver TG (left) and TC (right), relative mRNA levels of lipogenetic and uptake genes (H), proinflammatory genes (I), and profibrotic gene (J) in livers, (K) GTT, (L) ITT, (M) the AUC quantification of GTT and ITT from Thrsp -Flox and Thrsp -HKO mice fed with custom WD (modified western diet containing 40% kcal from fat and 0.2% cholesterol along with a high fructose–glucose solution) for 16 weeeks. (N) The serum MIF contents of Thrsp -Flox and Thrsp -HKO mice. (O, P) Flow cytometry analysis of liver monocytes stained with BODIPY-C16 (O) and CD74 (P), respectively, in Thrsp -Flox and Thrsp -HKO mice. (Q) Representative IHC staining (scale bar, 20 μm) and (Q) mIHC images (scale bar, 50 μm) in Thrsp -Flox and Thrsp -HKO mice. (R) The quantification of Q. Data were presented as the mean±SEM. All statistical analyses were carried out by an unpaired t test. * p <0.05, ** p <0.01, and *** p <0.001. Abbreviations: GTT, glucose tolerance test; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ITT, insulin tolerance test; LAMs, lipid-associated macrophages; MASH, metabolic dysfunction–associated steatohepatitis; MIF, macrophage migration inhibitory factor; mIHC, multiplex immunohistochemistry; ORO, oil red O; PA, palmitic acid; SDS, periportal (PP) marker gene; TC, total cholesterol; TG, triglycerides; Thrsp-HKO, thrsp knockout; WD, western diet.

Article Snippet: Subsequently, they were stained using the ORO stain kit by the manufacturer’s protocols (Sangon Biotech)., The primers for Thrsp overexpression plasmid construction and siRNA target sequences for Thrsp knockdown were listed in Supplemental Tables S4, http://links.lww.com/HEP/J840 , and S6, http://links.lww.com/HEP/J840 .

Techniques: Expressing, Staining, Modification, Western Blot, Flow Cytometry, Immunohistochemistry, Migration, Multiplex Assay, Marker, Knock-Out