Journal: Journal of pharmacological sciences

Article Title: Farrerol suppresses osteoclast differentiation and postmenopausal osteoporosis by inhibiting the nuclear factor kappa B signaling pathway.

doi: 10.1016/j.jphs.2023.12.011

Figure Lengend Snippet: Fig. 6. Histological and immunohistochemical analyses of distal femur sections from mice. (A) Representative images of H&E, Masson’s trichrome, and TRAP staining. The trabecular bone was sparser in OVX mice, but the density improved in response to treatment with 25 and 50 mg/kg farrerol. Images were scanned and presented at low magnification ( × 2 magnification, scale bar, 500 μm) and high magnification ( × 20 magnification, scale bar, 50 μm). (B) Quantification of TRAP staining. Farrerol treatment (25 and 50 mg/kg) effectively prevented in vivo osteoclast formation. (C) Serum CTX-1 levels. TRAP-positive multinucleated osteoclasts in the trabecular bone region immediately below the whole growth plate were evaluated. Results of TRAP staining and ELISA were obtained from six independent experiments. (D) Representative images of the immunohistochemical staining of p-P65 and p-P38 ( × 40 magnification, scale bar, 25 μm). Quantitative analysis of OCN expression levels were obtained from 10 fields from six independent experiments. **P < 0.01. n.s., not significant.

Article Snippet: The slices were evaluated using hematoxylin and eosin (H&E), Masson’s trichrome, and TRAP staining, as reported previously.11 For IHC analyses, the slices were treated overnight with primary antibodies against OCN (1:500; 23418-1-AP, Proteintech), p-P65 (1:50; ab31473, Abcam) or p-P38 (1:100; #4511, Cell Signaling Technology) at 4 ◦C.

Techniques: Immunohistochemical staining, Staining, In Vivo, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Journal of pharmacological sciences

Article Title: Farrerol suppresses osteoclast differentiation and postmenopausal osteoporosis by inhibiting the nuclear factor kappa B signaling pathway.

doi: 10.1016/j.jphs.2023.12.011

Figure Lengend Snippet: Fig. 7. Farrerol did not affect osteoblastic bone formation in vitro or in vivo. (A) Representative ALP staining images (scale bar, 500 μm). (B) Representative ARS staining images (scale bar, 500 μm). (C) Quantitative analysis of ALP-positive areas. (D) Quantitative analysis of ARS-positive areas. (E) Representative images of the immunohistochemical staining of OCN. Images were scanned and presented at low magnification ( × 2 magnification, scale bar, 500 μm) and high magnification ( × 20 magnification, scale bar, 50 μm). (F) Quantitative analysis of OCN expression levels. (G) Serum P1NP level. (H) Representative images of calcein-alizarin red S double labeling (scale bar, 20 μm). Compared to that in the OVX group, farrerol did not affect the MS/BS (I) or MAR (J). The results of ALP and ARS staining were obtained from nine randomly selected visual fields from three independent experiments. In the in vivo study, data were obtained from 18 fields from six independent experiments. *P < 0.05. n.s., not significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The slices were evaluated using hematoxylin and eosin (H&E), Masson’s trichrome, and TRAP staining, as reported previously.11 For IHC analyses, the slices were treated overnight with primary antibodies against OCN (1:500; 23418-1-AP, Proteintech), p-P65 (1:50; ab31473, Abcam) or p-P38 (1:100; #4511, Cell Signaling Technology) at 4 ◦C.

Techniques: In Vitro, In Vivo, Staining, Immunohistochemical staining, Expressing, Labeling

Journal: Frontiers in cell and developmental biology

Article Title: miR-129-5p Inhibits Bone Formation Through TCF4.

doi: 10.3389/fcell.2020.600641

Figure Lengend Snippet: FIGURE 6 | Rescue effect of miR-129-5p inhibitor on postmenopausal osteoporosis. (A) Representative images showing calvarial mineral apposition rate of C57BL/6 mice after OVX and inhibitor-129-5p treatment. Scale bar: 10 µm. BL (baseline): sacrifice before RNA treatment. Sham: sham OVX operation group. OVX: OVX group. Mock: transfection reagent control group. inhibit-NC: inhibitor-NC–treated group. inhibit-129: inhibitor-129-5p–treated group. (B) Calvarial mineral apposition rates of C57BL/6 mice after OVX and inhibitor-129-5p treatment (mean ± SD, ∗∗∗P < 0.001). (C) Expression of OCN in calvarial tissues of C57BL/6 mice after OVX and inhibitor-129-5p treatment, as detected by immunohistochemical staining. Scale bar: 50 µm. (D) Quantification of relative integrated optical density (IOD) values of OCN immunostaining using Image-Pro Plus 6.0 software (mean ± SD, ∗∗∗P < 0.001). (E) Expression of OXTERIX in calvarial tissues of C57BL/6 mice after OVX and inhibitor-129-5p treatment, as detected by immunohistochemical staining. Scale bar: 50 µm. (F) Quantification of relative IOD values of OXTERIX immunostaining using Image-Pro Plus 6.0 software (mean ± SD, ∗∗∗P < 0.001). (G) Expression of RUNX2 in calvarial tissues of C57BL/6 mice after OVX and inhibitor-129-5p treatment, as detected by immunohistochemical staining. Scale bar: 50 µm. (H) Quantification of relative IOD values of RUNX2 immunostaining using Image-Pro Plus 6.0 software (mean ± SD, ∗∗∗P < 0.001).

Article Snippet: Sections (5 μm in thickness) were dewaxed, immersed in the distilled water, blocked in 5% goat serum (CWBIO, CW0130) in PBS, and then incubated overnight at 4◦C with primary antibodies against OCN (Rabbit pAb, 1:200, Santa Cruz Biotech, sc365797, Dallas, TX, United States), OSTERIX (Rabbit pAb, 1:50; Proteintech, 12593-1-AP), and TCF4 (Rabbit pAb, 1:100, Proteintech, 22337-1-AP), respectively.

Techniques: Transfection, Control, Expressing, Immunohistochemical staining, Staining, Immunostaining, Software

Journal: Frontiers in cell and developmental biology

Article Title: miR-129-5p Inhibits Bone Formation Through TCF4.

doi: 10.3389/fcell.2020.600641

Figure Lengend Snippet: FIGURE 7 | miR-129-5p and osteogenic transcript factor expression levels of OVX mice after inhibitor-129-5p treatment. (A) miR-129-5p level in calvarial tissue of C57BL/6 mice after OVX and inhibitor-129-5p treatment, as detected by reverse transcriptase-polymerase chain reaction (RT-PCR; mean ± SD, ∗∗P < 0.01, ∗∗∗P < 0.001). BL (baseline): sacrifice before RNA treatment. Sham: sham OVX operation group. OVX: OVX group. Mock: transfection reagent control group. inhibit-NC: inhibitor-NC–treated group. inhibit-129: inhibitor-129-5p–treated group. (B–F) Ocn, Osterix, Tcf4, Tcf7, and Lef1 expression levels in calvarial tissue of C57BL/6 mice after OVX and inhibitor-129-5p treatment, as detected by RT-PCR (mean ± SD, ∗∗P < 0.01, ∗∗∗P < 0.001).

Article Snippet: Sections (5 μm in thickness) were dewaxed, immersed in the distilled water, blocked in 5% goat serum (CWBIO, CW0130) in PBS, and then incubated overnight at 4◦C with primary antibodies against OCN (Rabbit pAb, 1:200, Santa Cruz Biotech, sc365797, Dallas, TX, United States), OSTERIX (Rabbit pAb, 1:50; Proteintech, 12593-1-AP), and TCF4 (Rabbit pAb, 1:100, Proteintech, 22337-1-AP), respectively.

Techniques: Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Transfection, Control

Journal: Journal of Biological Engineering

Article Title: Designing of PLA scaffolds for bone tissue replacement fabricated by ordinary commercial 3D printer

doi: 10.1186/s13036-017-0074-3

Figure Lengend Snippet: Confocal microscopy photomicrographs of ST1 and ST2 seeded with osteosarcoma cells. Confocal microscopy photomicrographs of ST1 ( a , c ) and ST2 ( b , d ) scaffolds from polylactic acid seeded with osteosarcoma cells MG-63 after a 7-day and 14-day culture. Immunohistochemical staining using monoclonal antibody against either type I collagen ( a , b ) or osteocalcin ( c , d ), followed by secondary antibody conjugated with Alexa Fluor 488® ( green ) and propidium iodide staining of cell nuclei ( red ) showed groups of cells producing type I collagen on both scaffolds ( a , b ) after 7 days, but only rare osteocalcin staining in both scaffolds ( c , d ) after 14 days. Objective ×10×, magnification ×4, bar = 20 μm

Article Snippet: Immunohistochemical staining was performed using mouse monoclonal antibody against osteocalcin (overnight, 2–8 °C, dilution 1:200, ab13420, Abcam) or mouse monoclonal antibody against type I collagen (concentrate, overnight, 2–8 °C, dilution 1:20, clone M-38c was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242) and subseaquently with secondary anti-mouse antibody conjugated with AlexaFluor® 488 (45 min RT, dilution 1:300, A10667, Life Technologies).

Techniques: Confocal Microscopy, Immunohistochemical staining, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Spontaneous calcification process in primary renal cells from a medullary sponge kidney patient harbouring a GDNF mutation

doi: 10.1111/jcmm.12514

Figure Lengend Snippet: Immunocytochemistry for osteoblastic markers of MSK and control mesenchymal cells. Light microscopy images (200×) of MSK cells at p1 and p4 and of mesenchymal control cells at p4, showing 100% positivity for osteonectin; 40% of the cells were faintly positive for osteocalcin. Osteopontin was expressed by 100% of MSK cells at p1 and by 60% of mesenchymal cells (localized around the nuclei). Note the different localization of Runx2 immunopositivity, which was nuclear in MSK cells at p1 and in mesenchymal cells at p4, whereas Runx2 immunopositivity was surprisingly cytoplasmic in MSK cells at p4. The figures and percentages are representative of the results of three experiments.

Article Snippet: The cells were stained with monoclonal and polyclonal antibodies against osteocalcin (QED Bioscience San Diego, CA, USA), osteonectin (Chemicon), osteopontin (Chemicon) and Runx2 (Abnova Taipei, Taiwan), and the slides were processed using the same procedure described for immunophenotyping.

Techniques: Immunocytochemistry, Control, Light Microscopy