Journal: bioRxiv

Article Title: Estrogen Receptor 1 Signaling in Hepatic Stellate Cells Designates Resistance to Liver Fibrosis

doi: 10.1101/2024.10.10.617541

Figure Lengend Snippet: C57BL/6 wild-type female mice of 6 weeks old underwent ovariectomy or sham surgery. The mice were then fed with the normal chow diet (NCD) or methionine-choline deficient (MCD) diet to induce liver fibrosis. (A) Diagram of the experimental procedure. (B to F) Paraffin sections of the liver tissues of indicated conditions were assessed by histochemistry or immunohistochemistry. (B) Representative images of H&E staining. (C and D) Representative images of Sirius Red staining (C) and the quantification of the percentage (%) of Sirius Red-positive area (D) . (E and F) Representative images of anti-α-SMA immunohistochemistry (E , black arrows exemplify anti-α-SMA signals ) and the quantification of the percentage (%) of α-SMA-positive area (F) . mean ± SEM, * p < 0.05, ** p < 0.01 (Student’s t -test). (G and H) Cryosections of the liver tissues were examined by the immunofluorescence co-staining of TIMP1 and α-SMA. (G) Representative images were shown. White arrowheads exemplify anti-TIMP1 signals. (H) The mean fluorescence intensity of anti-TIMP1 signals was quantified. mean ± SEM, * p < 0.05 (Student’s t -test). (I) mRNA levels of fibrosis-related genes were determined by the qPCR analyses. mean ± SEM, * p < 0.05, n.s., not significant (two-way ANOVA test).

Article Snippet: For the immunofluorescence staining, 10-μm cryosections were blocked with PBS / 3% BSA and immunostained with the intended primary antibodies, including rabbit anti-ESR1 antibody (Millipore, #06-935), rabbit anti-ESR2 antibody (Invitrogen, #PA1311), goat anti-α-SMA antibody (Novus, #NB300-978), rabbit anti-TIMP1 antibody (Bioss, #bs-0415R), and chicken anti-GFP antibody (Aves Labs, #GFP-1010).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Estrogen Receptor 1 Signaling in Hepatic Stellate Cells Designates Resistance to Liver Fibrosis

doi: 10.1101/2024.10.10.617541

Figure Lengend Snippet: C57BL/6 wild-type male mice of 4 weeks old underwent castration and 17β-estradiol (β-E2) hormone replacement. The mice then received the normal chow diet (NCD) or methionine-choline deficient (MCD) diet to induce liver fibrosis. (A) Diagram of the experimental procedure. (B to F) Paraffin sections of the liver tissues of indicated conditions were assessed by histochemistry or immunohistochemistry. (B) Representative images of H&E staining. (C and D) Representative images of Sirius Red staining (C) and the quantification of the percentage (%) of Sirius Red-positive area (D) . (E and F) Representative images of anti-α-SMA immunohistochemistry (E , black arrows exemplify anti-α-SMA signals ) and the quantification of the percentage (%) of α-SMA-positive area (F) . mean ± SEM, * p < 0.05, ** p < 0.01 (Student’s t -test). (G and H) Cryosections of the liver tissues were examined by the immunofluorescence co-staining of TIMP1 and α-SMA. (G) Representative images were shown. White arrowheads exemplify anti-TIMP1 signals. (H) The mean fluorescence intensity of anti-TIMP1 signals was quantified. mean ± SEM, * p < 0.05 (Student’s t -test). (I) mRNA levels of fibrosis-related genes were determined by the qPCR analyses. mean ± SEM, ** p < 0.01, n.s., not significant (two-way ANOVA test).

Article Snippet: For the immunofluorescence staining, 10-μm cryosections were blocked with PBS / 3% BSA and immunostained with the intended primary antibodies, including rabbit anti-ESR1 antibody (Millipore, #06-935), rabbit anti-ESR2 antibody (Invitrogen, #PA1311), goat anti-α-SMA antibody (Novus, #NB300-978), rabbit anti-TIMP1 antibody (Bioss, #bs-0415R), and chicken anti-GFP antibody (Aves Labs, #GFP-1010).

Techniques: Immunohistochemistry, Staining, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Estrogen Receptor 1 Signaling in Hepatic Stellate Cells Designates Resistance to Liver Fibrosis

doi: 10.1101/2024.10.10.617541

Figure Lengend Snippet: (A) HSCs in the liver tissues of Lrat Cre ; RCL-ChR2(H134R)/EYFP +/- mice fed with the normal chow diet were examined by FACS. A vast majority (>99%) of retinol + CD45 - HSCs were EYFP-positive. (B to J) Lrat Cre ;Esr1 fl/fl or control Esr1 fl/fl female littermates of 8 weeks old received the normal chow diet (NCD) or methionine-choline deficient (MCD) diet to induce liver fibrosis. (B) HSCs were FACS-sorted from the liver tissues of Lrat Cre ;Esr1 fl/fl or control Esr1 fl/fl mice. Genetic deletion of Esr1 in Lrat Cre ;Esr1 fl/fl HSCs was validated by the qPCR analysis. mean ± SEM, ** p < 0.01 (Student’s t -test). (C to G) Paraffin sections of the liver tissues of indicated conditions were assessed by histochemistry or immunohistochemistry. (C) Representative images of H&E staining. (D and E) Representative images of Sirius Red staining (D) and the quantification of the percentage (%) of Sirius Red-positive area (E) . (F and G) Representative images of anti-α-SMA immunohistochemistry (F , black arrows exemplify anti-α-SMA signals ) and the quantification of the percentage (%) of α-SMA-positive area (G) . mean ± SEM, * p < 0.05, ** p < 0.01 (Student’s t -test). (H and I) Cryosections of the liver tissues were examined by the immunofluorescence co-staining of TIMP1 and α-SMA. (H) Representative images were shown. White arrowheads exemplify anti-TIMP1 signals. (HI The mean fluorescence intensity of anti-TIMP1 signals was quantified. mean ± SEM, * p < 0.05 (Student’s t -test). (I) mRNA levels of fibrosis-related genes were determined by the qPCR analyses. mean ± SEM, * p < 0.05, n.s., not significant (two-way ANOVA test).

Article Snippet: For the immunofluorescence staining, 10-μm cryosections were blocked with PBS / 3% BSA and immunostained with the intended primary antibodies, including rabbit anti-ESR1 antibody (Millipore, #06-935), rabbit anti-ESR2 antibody (Invitrogen, #PA1311), goat anti-α-SMA antibody (Novus, #NB300-978), rabbit anti-TIMP1 antibody (Bioss, #bs-0415R), and chicken anti-GFP antibody (Aves Labs, #GFP-1010).

Techniques: Control, Immunohistochemistry, Staining, Immunofluorescence, Fluorescence

Journal: International Journal of Nephrology

Article Title: Effect of the Direct Renin Inhibitor Aliskiren on Urinary Albumin Excretion in Spontaneous Type 2 Diabetic KK- A y Mouse

doi: 10.1155/2013/519130

Figure Lengend Snippet: Expression of MMP-2, MMP-9, TIMP-1, TIMP-2, fibronectin, type IV collagen, MCP-1, and (P)RR in the kidneys of each mouse using real-time PCR (A: untreated KK mice at 8 weeks of age, B: untreated KK mice at 12 weeks of age, C: untreated KK- A y mice at 8 weeks of age, D: untreated KK- A y mice at 12 weeks of age, and E: treated KK- A y mice at 12 weeks of age). The ratio of MMP-2 (a), MMP-9 (b), TIMP-1 (c), TIMP-2 (d), fibronectin (e), type IV collagen (f), MCP-1 (g), and (P)RR (h) mRNAs to GAPDH mRNA is shown in the kidneys of mice from each group. These expressions were increased significantly in group 4 compared with groups 1, 2, and 3. This increase was attenuated in group 5 (* P < 0.001 versus untreated KK- A y mice at 12 weeks of age).

Article Snippet: The primary antibodies (Abs) were as follows: goat polyclonal anti-MMP-2 Ab (R&D Systems, Inc., Minneapolis, MN, USA), goat polyclonal anti-MMP-9 Ab (R&D Systems, Inc.), rabbit polyclonal anti-TIMP-1 Ab (Abbiotec, LLC, San Diego, CA, USA), rabbit polyclonal anti-TIMP-2 Ab (Abbiotec, LLC), and rat monoclonal anti-F4/80 Ab (MAC497GA; Serotec, Oxford, UK).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: International Journal of Nephrology

Article Title: Effect of the Direct Renin Inhibitor Aliskiren on Urinary Albumin Excretion in Spontaneous Type 2 Diabetic KK- A y Mouse

doi: 10.1155/2013/519130

Figure Lengend Snippet: Immunohistochemical staining of MMP-2, MMP-9, TIMP-1, and TIMP-2 in the renal sections of each mouse (O: untreated KK- A y mice at 12 weeks of age without primary Ab, A: untreated KK mice at 8 weeks of age, B: untreated KK mice at 12 weeks of age, C: untreated KK- A y mice at 8 weeks of age, D: untreated KK- A y mice at 12 weeks of age, and E: treated KK- A y mice at 12 weeks of age). Stainings showing the expression of MMP-2 (a), MMP-9 (b), TIMP-1 (c), and TIMP-2 (d) in the kidneys of mice from each group. These expressions were increased significantly in group 4 compared with groups 1, 2, and 3. This increase was attenuated in group 5 (* P < 0.001 versus untreated KK- A y mice at 12 weeks of age). Images were taken at 400-fold (a and d) and 200-fold (b and c) magnification. WGA indicates whole glomerular area. WIA indicates whole interstitial area.

Article Snippet: The primary antibodies (Abs) were as follows: goat polyclonal anti-MMP-2 Ab (R&D Systems, Inc., Minneapolis, MN, USA), goat polyclonal anti-MMP-9 Ab (R&D Systems, Inc.), rabbit polyclonal anti-TIMP-1 Ab (Abbiotec, LLC, San Diego, CA, USA), rabbit polyclonal anti-TIMP-2 Ab (Abbiotec, LLC), and rat monoclonal anti-F4/80 Ab (MAC497GA; Serotec, Oxford, UK).

Techniques: Immunohistochemical staining, Staining, Expressing