rabbit anti-human col1 antibody  (Thermo Fisher)

Journal: Stem Cell Research & Therapy

Article Title: Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects

doi: 10.1186/s13287-021-02580-7

Figure Lengend Snippet: IL-1β priming allows successful fracture repair. Upon IL-1β treatment, a trend towards a decrease in chondromodulin was detected, while IL-1β upregulated VEGF and MMP13 ( A ). Safranin-O staining prior to implantation further revealed the increase in chondrocyte lacunae size, suggesting progressive maturation towards chondrocyte hypertrophy ( B ). Following orthotopic implantation, the cartilaginous organoids underwent progressive mineralization with bone bridging being detected in the µCT images after 4 weeks ( C ). µCT quantification revealed a significant increase ( p = 0.0095) in the volume of mineralized tissue in the defect area between BBT3 and BBT3 + IL-1β conditions after 8 weeks ( D ). Histological analysis using safranin-O further revealed accelerated bone formation with cartilage resorption being detected at week 4 ( E ). While bone union was detected after 8 weeks, some cartilaginous remnants could be detected, which indicates progressive cartilage resorption. Immunostaining of human collagen type I further revealed that the newly formed bone was host derived, while some hypertrophic chondrocytes expressed low levels of collagen type I ( F ). Mathematical modelling was used to confirm the cumulative effects of BMP, Wnt, TH and IL-1β in chondrocyte hypertrophy. An increase in hypertrophy chance, along with a decrease in variability, was detected following in vitro stimulation with aforementioned factors. DC: β-catenin Destruction Complex ( G ). The presented data have been collected from experiments ( n = 6) using CY2 cell line. Statistical significance is represented as follow: * p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001. The following abbreviations have been used to highlight the tissue composition in the sections: B: bone, C: cartilage, M: bone marrow, T: cartilage-bone turnover site (dotted rectangle) and CR: cartilage remnants

Article Snippet: Sections were then washed with PBS + 1% Tween 20 and blocked with 5% bovine serum albumin (BSA) for 30 min (Sigma, UK), before being incubated with a rabbit anti-human Col1 antibody (1:200, Thermo Scientific, USA) at 4 °C overnight.

Techniques: Staining, Immunostaining, Derivative Assay, In Vitro

Journal: Mucosal immunology

Article Title: Interleukin (IL)-4, IL-13, and IL-17A differentially affect the profibrotic and proinflammatory functions of fibrocytes from asthmatic patients.

doi: 10.1038/mi.2011.60

Figure Lengend Snippet: Figure 1 Comparison of the frequency of CD34 + COL1 + ( a ) and CD34 + COL1A1 mRNA + fibrocytes ( b ) in the peripheral blood of healthy controls and asthmatic patients. The percentage of cells coexpressing CD34 and COL1 or CD34 and COL1A1 mRNA was calculated on cytospins of total peripheral blood leukocytes. The horizontal lines indicate the medians. * * * P < 0.001 vs. healthy controls by the Mann – Whitney test.

Article Snippet: For intracellular staining of COL1, cells were permeabilized with 0.3 % saponin solution (Sigma-Aldrich, St Louis, MO) in Tris-buffered saline and sequentially 148 VOLUME 5 NUMBER 2 | MARCH 2012 | www.nature.com/mi incubated with a rabbit anti-human COL1 ( 2 chain) (Novus Biologicals, Littletone, CO), a biotinylated goat anti-rabbit secondary antibody (Novus Biologicals), and rhodamine red X-conjugated streptavidin (Jackson ImmunoResearch, Bar Harbor, ME).

Techniques: Comparison, MANN-WHITNEY

Journal: Mucosal immunology

Article Title: Interleukin (IL)-4, IL-13, and IL-17A differentially affect the profibrotic and proinflammatory functions of fibrocytes from asthmatic patients.

doi: 10.1038/mi.2011.60

Figure Lengend Snippet: Figure 2 Isolation and phenotypic analysis of circulating fibrocytes. CD34 + cells were isolated from the peripheral blood by positive immunomagnetic selection and cultured for 5 days to obtain a pure population of mature fibrocytes. The non-adherent cells were removed after the first 48 h and the adherent cells were reincubated for the next 3 days in fresh medium. Flow cytometric analysis of the isolated cells was performed soon after the immunomagnetic selection ( a ) and on day 5 ( c ). The horizontal and vertical lines in the representative dot plots mark fluorescence intensity greater than that observed with isotype-matched controls for the anti-CD45, anti-CD34, and anti-COL1 antibodies. The morphology of the cells in culture was monitored under an inverted microscope and the microphotographs in panel b show representative images in phase-contrast mode taken at the indicated points in time. The expression of the type I interleukin (IL)-4 receptor subunits (IL-4R / common -chain ( c)), type II IL-4 receptor subunits (IL-4R / IL-13R 1), IL-13R 2, and IL-17RA on the membrane of control and asthmatic fibrocytes were evaluated by flow cytometry on day 5 ( d ). The relative level of expression was quantified by subtracting the median fluorescence intensity (MFI) of cells stained with each specific antibody from the MFI of cells stained with the corresponding isotype control and dividing the obtained value by the MFI of unstained cells. Data are expressed as the means and s.d. * P < 0.05 vs. control fibrocytes; * * P < 0.01 vs. control fibrocytes by the unpaired Student ’ s t -test; n = 5.

Article Snippet: For intracellular staining of COL1, cells were permeabilized with 0.3 % saponin solution (Sigma-Aldrich, St Louis, MO) in Tris-buffered saline and sequentially 148 VOLUME 5 NUMBER 2 | MARCH 2012 | www.nature.com/mi incubated with a rabbit anti-human COL1 ( 2 chain) (Novus Biologicals, Littletone, CO), a biotinylated goat anti-rabbit secondary antibody (Novus Biologicals), and rhodamine red X-conjugated streptavidin (Jackson ImmunoResearch, Bar Harbor, ME).

Techniques: Isolation, Selection, Cell Culture, Fluorescence, Inverted Microscopy, Expressing, Membrane, Control, Flow Cytometry, Staining