Journal: Scientific Reports

Article Title: Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration

doi: 10.1038/s41598-022-18675-x

Figure Lengend Snippet: The histomorphological and immunohistochemical features of the bone-like tissues. ( a ) HE staining showed osteoblast-like cells surrounded by a cohesive osteoid-like matrix and osteocyte-like cells embedded into lacunae-like structures (black arrowheads) in the 3:1, 2:1, and 1:1 co-cultures. ( b , c ) Osteoblast-like cells on the osteoid-like matrix surface were positive for ALP (blue arrowheads) and Col1 (orange arrowheads). ( d ) Osteocyte-like cells in the osteoid-like matrix were positive for DMP1 (green arrowheads). ( e , f ) Fluorescence microscopy showed that osteocyte-like cells embedded in the osteoid-like matrix and osteoblast-like cells on the surface were GFP positive (red arrowheads). DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: After a treatment with 0.1% trypsin for 30 min, the sections of bone-like tissues were incubated at 4 °C overnight with a Col1 antibody (NB600-408, 1:100, Novus Biologicals, Littleton, CO, USA) and a DMP1 antibody (sc-54181, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with secondary antibodies.

Techniques: Immunohistochemical staining, Staining, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: Spheroid co-culture of BMSCs with osteocytes yields ring-shaped bone-like tissue that enhances alveolar bone regeneration

doi: 10.1038/s41598-022-18675-x

Figure Lengend Snippet: Transplantation of bone-like tissue in a tooth-extraction mouse model. ( a ) The extraction sockets in the bone-like tissue implantation group showed better healing (red box area). Micro-CT ( b , c ) and HE staining (red box area) ( c ) of the extraction sockets showed that the bone mass in the bone-like tissue implantation group was higher than in unimplanted controls. BV/TV, bone volume/total volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation. ( d ) The extraction sockets of the bone-like tissue implantation group had more osteoid and mineralized bone by Goldner’s trichrome staining (red box area). Ordinary white-light microscopy ( e ) and polarized light microscopy ( f ) showed picrosirius red staining for the collagen fibers in the extraction sockets. ( g , h ) The bone-like tissue implantation group had more total collagen and type I collagen (red) than unimplanted controls. * P < 0.05.

Article Snippet: After a treatment with 0.1% trypsin for 30 min, the sections of bone-like tissues were incubated at 4 °C overnight with a Col1 antibody (NB600-408, 1:100, Novus Biologicals, Littleton, CO, USA) and a DMP1 antibody (sc-54181, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with secondary antibodies.

Techniques: Transplantation Assay, Extraction, Micro-CT, Staining, Light Microscopy

Journal: Bioengineering

Article Title: All Trans-Retinoic Acids Facilitate the Remodeling of 2D and 3D Cultured Human Conjunctival Fibroblasts

doi: 10.3390/bioengineering9090463

Figure Lengend Snippet: Effects of ATRA toward the gene expression of ECMs of the TGF-β2 untreated or treated HconF cells. Two-dimensional and three-dimensional HconF cells untreated and treated with TGF-β2 (TGFβ, 5 ng/mL) were subjected to qPCR analysis in the presence or absence of 1 μM ATRA and the mRNA expressions in ECMs, including COL1 , COL4 , COL6 , FN, and aSMA, were evaluated. All experiments were performed in triplicate using 3 different confluent 6-well dishes (2D) or 15 freshly prepared 3D HconF spheroids (3D) in each experimental condition. Data are expressed as the mean ± standard error of the mean (SEM). * p < 0.05, ** p < 0.01 and *** p < 0.005; ANOVA followed by a Tukey’s multiple comparison test.

Article Snippet: They were then sequentially treated with (1) 1:200 dilutions of primary antibodies; an anti-human COL1 (#600-401-103-0.1, ROCKLAND Antibodies & Assays, Limerick, PA, USA), COL4 (#600-401-106S, ROCKLAND Antibodies & Assays, Limerick, PA, USA), COL6 (#009-001-108, ROCKLAND Antibodies & Assays, Limerick, PA, USA), or FN (#A-11, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) rabbit antibody at 4 °C overnight, (2) washed 3 times with PBS for 1 h each, (3) 1:1000 dilutions of a secondary antibody; a goat anti-rabbit IgG (488 nm, #A27034, Invitrogen, Waltham, MA, USA) with phalloidin (594 nm, 1:1000 dilutions, # 17466-45-4, Cayman Chemical, Ann Arbor, MI, USA) and DAPI (1:1000 dilutions, # D523-10, DOJINDO, Osaka, Japan) for 3 hrs, and (4) mounting ProLong Gold Antifade Mountant with a cover glass.

Techniques: Gene Expression, Comparison

Journal: Cells

Article Title: Reciprocal Regulation of TRPS1 and miR-221 in Intervertebral Disc Cells

doi: 10.3390/cells8101170

Figure Lengend Snippet: Evaluation of the effect of TRPS1 overexpression on IVD cells. Cells were transfected with the vector containing the full-length human TRPS1 cDNA (black column), with the empty vector (gray column) or remained untreated (CTR, control, white column) and harvested after 48 h. The expression of differentiation markers ( A ), miR-221 ( B ), antioxidant defense and stemness markers ( C ) were evaluated. Representative optical photomicrographs of TRPS1, COL2, SOX9, ACAN, COL1, SOD2 and SOX-2 respectively immunostaining is reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software and expressed as means of pixels per one hundred cells ± SD ( n = 10). * = p < 0.01. Scale bars: 20 µm. The expression levels of miR-221 were measured by qRT-PCR by the 2 (−△△Ct) method. Data are presented as fold changes relative to control untreated cells ± SD ( n = 10). * = p < 0.01 (hTRPS1 treatment vs. CTR and empty).

Article Snippet: After the incubation in blocking serum, the different primary antibodies were added and incubated at 4 °C overnight: Polyclonal antibodies for COL2A1 (#Ab3092; mouse anti-human, 1:200 dilution, Abcam, Cambridge, UK), SOX9 (#sc-20095; rabbit anti-human, 1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) ACAN (#sc-33695; mouse anti-human, 1:200 dilution, Santa Cruz Biotechnology), TRPS1 (#20003-1-AP; rabbit anti-human, 1:100 dilution; Proteintech Group, Rosemont, WA, USA), COL1 (#sc-28657; rabbit anti-human, 1:200 dilution, Santa Cruz Biotechnology), SOX-2 (#sc-365823; mouse anti-human, 1:500 dilution, Santa Cruz Biotechnology) and SOD2 (#sc-133134; mouse anti-human; 1:200 dilution; Santa Cruz Biotechnology).

Techniques: Over Expression, Transfection, Plasmid Preparation, Control, Expressing, Immunostaining, Software, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Melatonin Exerts Prominent, Differential Epidermal and Dermal Anti-Aging Properties in Aged Human Eyelid Skin Ex Vivo

doi: 10.3390/ijms242115963

Figure Lengend Snippet: List of antibodies and immunohistochemical staining treatments used in the study.

Article Snippet: Collagen I α-1 , Acetone, 10 min at −20 °C , 10% goat serum in PBS , mouse monoclonal anti-Collagen I alpha-1 (1:500; Novus Bio, #NB600-450) , goat monoclonal anti-mouse IgG-Alexa Fluor 488 (1:400; Invitrogen, A11001).

Techniques: Immunohistochemical staining, Staining, Blocking Assay, Amplification, Avidin-Biotin Assay