rabbit anti collagen xii  (Bio-Rad)

Journal: Scientific Reports

Article Title: Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis

doi: 10.1038/s41598-024-59713-0

Figure Lengend Snippet: Immunocytochemical features of gingival connective tissues in the gingiva of RNS variants. ( A ) In normal gingiva, POSTN is localized in a loose extracellular network around blood vessels (arrowheads; bv) whereas in both RNS-1 ( B ) and RNS-2 ( C ) mutants POSTN extracellular expression is localized along thick and disorganized fibers in the connective tissue (ct). ( D ) In normal gingiva, Collagen 5 alpha-1 (green; Col5A1) is secreted and deposited along collagen 1 fibers and Vimentin (red; Vim) is expressed by endothelial cells. ( E , F ) Increased expression of Col5A1 is detected in the connective tissue (ct) of RNS-1 ( E ) and RNS-2 ( F ) mutants.Vim is normally localized in endothelial cells and abnormally in fibroblasts ( F ). ( G ) Alpha-SMA (α-SMA) is a specific marker of contractile endothelial cells of bv. ( H , I ) In RNS mutants, α-SMA is also expressed by fibroblasts in ct. Note the presence of inflammatory infiltrates (i) in RNS-1 gingiva. ( J ) In normal gingiva, Fibroblast-specific protein 1 (green; FSP1) is expressed by gingival fibroblasts (green arrow). Osteonectin (red; SPARC) is expressed in discrete spots by fibroblasts (red arrowhead) and endothelial cells. ( K , L ) FSP-1 is normally expressed in ct. SPARC expression and SPARC deposition along fibers are dramatically increased in RNS mutant connective tissues. ( M ) Fibronectin (green; FN) is specifically expressed by fibroblasts. Phospho-SMAD3 (red; pSMAD3) is mainly localized in nuclei of vascular cells (arrowhead). ( N , O ) In RNS-derived gingiva FN expression is dramatically decreased. pSMAD3 expression is dramatically increased in both vascular cells (arrowhead) and fibroblasts (arrows) nuclei. Scale bars: ( A – C ), ( G – L ) = 100 μm; ( D – F ), ( M – O ) = 30 μm.

Article Snippet: Sections were incubated for 2 h at room temperature or overnight at 4 °C with primary antibodies; rabbit anti-collagen V alpha-1 (1/1000; NBP-38162; Novus biologicals), rabbit anti-FAM20A (1/250; OACD03385; Aviva), rabbit anti-Fam20C (1/250; OAAB01003; Aviva), rabbit anti-Periostin (1/500; ab14041; Abcam), rabbit anti-phospho-SMAD3 (1/500; ab52903; Abcam), rabbit anti-SPARC (1/2000; ab255733; ABCAM), mouse anti-alpha Smooth Muscle Actin (1/1000; 1A4; ab7817; Abcam), mouse anti-Fibronectin (1/1000; P1H11; DSHB), mouse anti-FSP1 (1/2000; ab218512; ABCAM), mouse anti-Vimentin (1/1000; RV203; NBP-19767; Novus biologicals) and rat anti-Procollagen type I (1/500; M-58; ab64409; Abcam), mouse anti-Ubiquitin (UCHL1; 1/400; 66230-1-Ig; Proteintech), goat anti-Lamin B1(1/200; sc6216), Concanavalin A-Alexa 633 conjugate (70 mg/ml; 2616059; Thermo Fischer).

Techniques: Expressing, Marker, Mutagenesis, Derivative Assay

Journal: Scientific Reports

Article Title: Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis

doi: 10.1038/s41598-024-59713-0

Figure Lengend Snippet: SMAD3 activation in untreated RNS GFs and treated control and RNS GFs. Immunocytochemical staining of control ( A , D ) and RNS ( B , C , E , F ) GFs cultured without TGFβ1 ( A – C ) or with 5 ng/ml TGFβ1 ( D – F ) for 6 h. Cells were fluorescently labeled for p-SMAD3 (green), nuclei (blue), and the specific F-actin marker, phalloidin (red). Co-localization of p-SMAD3 and nuclei indicated nuclear translocation of p-SMAD3 (green arrow). ( A ) In control untreated GFs, p-SMAD3 immunoreactivity is detected at low levels in some nuclei. ( B , C ) In RNS untreated GFs, p-SMAD3 was increased in intensity in all nuclei. ( D – F ) TGFβ1 induced an increase in p-SMAD3 nuclei in normal ( D ) and mutant GFs ( E , F ). ( G ) Western blot were performed on cell lysates. P-YAP (Ser 397) protein levels were decreased in RNS GFs without or with TGFβ1 compared to Control. TAZ protein levels were increased in RNS GFs compared to normal GFs. Densitometric analysis of Phospho-YAP and TAZ bands normalized to corresponding GAPDH bands. Data represent mean fold change in band intensity ± s.d. relative to GAPDH of 3 independent experiments in triplicates. Data were analyzed by one-way ANOVA with Bonferroni multiple comparisons test (**p < 0.01, ***p < 0.001). ( H ) Western blots were performed on cell lysates. Alpha-SMA protein levels were increased in controls GFs cultured with TGFβ1. Alpha-SMA protein levels were increased in RNS GFs cultured without or with TGFβ1 compared to control. Densitometric analysis of a-SMA bands normalized to corresponding GAPDH levels. Scale bars: ( A - F ) = 20 µm.

Article Snippet: Sections were incubated for 2 h at room temperature or overnight at 4 °C with primary antibodies; rabbit anti-collagen V alpha-1 (1/1000; NBP-38162; Novus biologicals), rabbit anti-FAM20A (1/250; OACD03385; Aviva), rabbit anti-Fam20C (1/250; OAAB01003; Aviva), rabbit anti-Periostin (1/500; ab14041; Abcam), rabbit anti-phospho-SMAD3 (1/500; ab52903; Abcam), rabbit anti-SPARC (1/2000; ab255733; ABCAM), mouse anti-alpha Smooth Muscle Actin (1/1000; 1A4; ab7817; Abcam), mouse anti-Fibronectin (1/1000; P1H11; DSHB), mouse anti-FSP1 (1/2000; ab218512; ABCAM), mouse anti-Vimentin (1/1000; RV203; NBP-19767; Novus biologicals) and rat anti-Procollagen type I (1/500; M-58; ab64409; Abcam), mouse anti-Ubiquitin (UCHL1; 1/400; 66230-1-Ig; Proteintech), goat anti-Lamin B1(1/200; sc6216), Concanavalin A-Alexa 633 conjugate (70 mg/ml; 2616059; Thermo Fischer).

Techniques: Activation Assay, Control, Staining, Cell Culture, Labeling, Marker, Translocation Assay, Mutagenesis, Western Blot

Journal: International journal of molecular sciences

Article Title: Anti-Fibrotic Effects of RF Electric Currents.

doi: 10.3390/ijms241310986

Figure Lengend Snippet: Figure 1. α-SMA, Col I and Col III expression. (A) α-SMA, Col I, and Col III protein expression of myofibroblasts at 48 h of CRET or sham treatment. Fluorescence intensity measurement per MHC channel. Data normalized over the corresponding sham-exposed controls (dashed line represents the

Article Snippet: At the end of 48 h RF- or sham-exposure, the samples were fixed with 4% paraformaldehyde (Merck, Darmstadt, Germany) and incubated overnight at 4 ◦C with mouse monoclonal anti- α-smooth muscle actin antibody α-SMA (1:400; cat. no. A 2547; Sigma Aldrich, St. Louis, MO, USA), rabbit polyclonal anti-collagen type I antibody (1:400; cat. no. NB600408-0.1 mg; Novus; Centennial, CO, USA) and rabbit polyclonal anti-collagen type III alpha 1 antibody (1:400, cat. no. NB600-594SS; Novus; CO, USA).

Techniques: Expressing, Fluorescence

Journal: bioRxiv

Article Title: Accelerated Bone Healing in Calvarial and Femoral Defects with Injectable Microcarriers that Mimic the Osteogenic Niche

doi: 10.1101/2021.11.05.467478

Figure Lengend Snippet: A) Schematic diagram of the steps to obtaining OCM-coated microcarriers. B-C: Representative low- and high-resolution SEM micrographs of GelMA (B, B’) and OCM-GelMA (C, C’) microcarriers. D-G) Representative immunofluorescence images of GelMA (D,F) and OCM-GelMA (E,G) microcarriers with antibodies against collagen VI (D, E) and XII (F, G).

Article Snippet: Microcarriers were incubated overnight at 4°C in blocking buffer with rabbit anti-human type VI collagen and rabbit anti-human type XII collagen primary antibodies (each 1:200, Novus Biologicals, Littleton, CO).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: Accelerated Bone Healing in Calvarial and Femoral Defects with Injectable Microcarriers that Mimic the Osteogenic Niche

doi: 10.1101/2021.11.05.467478

Figure Lengend Snippet: Effects of OCM and GW9662 on collagen and BMP-2 gene expression. Quantitative PCR was used to quantify the effects 10uM GW9662 and OCM on transcription of collagen VI (A) and collagen XII (B) on Day 8. (C) GW9662 and OCM-GelMA synergistically upregulated BMP-2 expression on Day 8. Data is presented as fold-change in gene expression relative to hMSC monolayers in Complete Culture Media. Statistics are (n=3) one sided ANOVA and Tukey post test ** represents p<0.005. All data presented as average ± s.d. All experiments performed in triplicate.

Article Snippet: Microcarriers were incubated overnight at 4°C in blocking buffer with rabbit anti-human type VI collagen and rabbit anti-human type XII collagen primary antibodies (each 1:200, Novus Biologicals, Littleton, CO).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing