C2456

Manufactured by Merck Group

Sourced in United States

The C2456 is a laboratory equipment product. It is designed for general laboratory use. The core function of the C2456 is to perform basic tasks essential for various laboratory procedures.

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Lab products found in correlation

22 protocols using c2456

Decellularization Effects on ECM Composition

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Direct and indirect immunofluorescence staining were performed on a 6 μm cryosection to evaluate the effect of decellularization on the ECM composition and to confirm the absence of nuclei. Native pericardium was used as the control. Before staining, samples were fixed in 4% (w/v) paraformaldehyde (PFA) supplied by Bioptica.
The primary antibodies used were collagen I (1:100, C2456; Sigma), elastin (1:50, ab21610, Abcam), and collagen IV (1:200, ab6586, Abcam). The controls were incubated with 1% (w/v) bovine serum albumin, instead of primary antibodies. In order to reveal the primary antibody binding, secondary antibodies were applied in separate: goat-antimouse Alexa Fluor 555 (1:300, A21422; Invitrogen) and goat anti-rabbit Alexa Fluor 555 (1:300, A27039; Invitrogen). Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), following the producer’s instructions. Filamentous actin was fluorescently labelled with Phalloidin–Atto 647N (1:200, 65906, Sigma-Aldrich, St. Louis, MO, USA).
Images were acquired with an epifluorescence microscope Leica AF6000, connected to a Leica DC300 digital camera and equipped with LAS AF Software (Leica Micro-System, Wetzlar, Germany). Image processing was performed using the open-source ImageJ software (NIH, Bethesda, MD, USA).

Todesco M., Imran S.J., Fortunato T.M., Sandrin D., Borile G., Romanato F., Casarin M., Giuggioli G., Conte F., Marchesan M., Gerosa G, & Bagno A. (2022). A New Detergent for the Effective Decellularization of Bovine and Porcine Pericardia. Biomimetics, 7(3), 104.

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Fibroblast Characterization after Genome Editing

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After genome editing, fibroblasts were left in culture for 1 week. Cells were then plated in a 96-well clear imaging microplates (BD Falcon) at a concentration of 1 × 10^{4 (link)} cells/well in DMEM (0.4% FBS). After 24 h, cells were incubated in modified medium containing 0.4% of FBS, 16.7 μg/mL of ascorbic acid, 37.5 mg/mL of Ficoll 70, and 25 mg/mL of Ficoll 400 to generate macromolecular crowding conditions. Transforming growth factor beta (TGF-β; 1 ng/mL) was also added, and fibroblasts were left at 37°C for 72 h before being fixed in ice-cold methanol and permeabilized in 0.1% Triton-X-100 in PBS. Immunostaining was performed by overnight incubation at 4°C with anti-collagen type I antibody (C2456; Sigma–Aldrich) at 1:1,000 dilution in PBS. Cells were then incubated for 1 h at room temperature with secondary antibody Alexa Fluor 488 goat anti-mouse immunoglobulin G (IgG; A11001; Invitrogen) at 1:500 dilution and Hoeschst dye (H3570; Invitrogen) at 1:10,000 dilution in PBS. A further staining step with anti-αSMA antibody (C6198; Sigma–Aldrich) at 1:1,000 dilution in PBS was carried out for 1 h at room temperature. The culture plate was scanned using a CellInsight NXT HCS instrument (Thermo Fisher Scientific) at 10 × magnification.

Martufi M., Good R.B., Rapiteanu R., Schmidt T., Patili E., Tvermosegaard K., New M., Nanthakumar C.B., Betts J., Blanchard A.D, & Maratou K. (2019). Single-Step, High-Efficiency CRISPR-Cas9 Genome Editing in Primary Human Disease-Derived Fibroblasts. The CRISPR Journal, 2(1), 31-40.

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Immunofluorescence Staining of Cellular Markers

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Cells were cultured on coverslips and were fixed with 4% paraformaldehyde for 20–30 min, permeabilized by 0.4% Triton X‐100, and blocked in 1% BSA/1x PBST at room temperature for 1 h. Primary and secondary antibodies were diluted in a ratio of 1:1000 and 1:10,000 with blocking buffer, respectively. The information of primary antibodies used in current study was listed as following: vimentin (ab24525, Abcam), α‐SMA (A2547, Sigma), collagen I (C2456, Sigma), FN1 (ED‐A) (ab6328, Abcam), MMP1 (ab137332, Abcam), MMP2 (GTX104577, GeneTex), MMP8 (GTX61732, GeneTex), MMP9 (ab38898, Abcam), and MMP13 (ab39012, Abcam). For secondary antibodies: anti‐rabbit Dylight 488 (SA5‐10038, Thermo Fisher Scientific, Waltham, MA), anti‐chicken Dylight 550 (SA5‐10071, Thermo Fisher Scientific), anti‐mouse Dylight 633 (35512, Thermo Fisher Scientific), and mouse Dylight 488 (35503, Thermo Fisher Scientific). Samples were immunostained with primary antibody (1:200) at 4°C overnight, and further incubated with secondary antibodies (1:200) conjugated with Dylight at room temperature for 1 h. Nuclei were counterstained with DAPI (1:1000), and the samples were mounted with anti‐fade solution. Fluorescent images were obtained using Leica TCS SP8 confocal microscope and analyzed by ImageJ software to determine the fluorescent intensity.⁷⁸, ⁷⁹, ⁸⁰

Hsiao Y., Wang I, & Yang T. (2022). Fibrotic remodeling and tissue regeneration mechanisms define the therapeutic potential of human muscular progenitors. Bioengineering & Translational Medicine, 8(2), e10439.

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Immunofluorescent Visualization of ECM Components

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The dECMs on tissue culture plates were fixed with 4% paraformaldehyde for 30 min. After blocking in 10% normal goat serum for 1 h, dECMs were incubated with monoclonal antibodies for type I collagen (clone COL-1, dilution 1:2000, catalog number C2456, Sigma-Aldrich) and fibronectin (EP5, dilution 1:200, catalog number sc-8422, Santa Cruz Biotechnology, Dallas, TX) overnight followed by Alexa Fluor 488 goat anti-mouse IgG (Life Technologies) for 30 min. dECMs were visualized with a Zeiss LSM 510 confocal on an AxioImager Z1 microscope using a 63× objective lens (Carl Zeiss, Jena, Germany).

Li J., Narayanan K., Zhang Y., Hill R.C., He F., Hansen K.C, & Pei M. (2019). Role of lineage-specific matrix in stem cell chondrogenesis. Biomaterials, 231, 119681.

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Characterization of Collagen Microspheres

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NBC-MSC-collagen microspheres were fixed in 4% PFA at room temperature in dark for 30 minutes and were dehydrated using a serial gradient ethanol treatment before processing for paraffin sections of 5μm thickness. Routine hematoxylin and eosin (Sigma) staining was conducted to reveal the cell morphology in the microspheres. To evaluate the presence of NBCs, a primary antibody (ab49501, abcam) was used. Anti-mouse secondary antibody (BA-1000, Vector laboratories) was used in immunohistochemistry, followed by ABC staining, diaminobenzidine labelling, and counterstaining using hematoxylin. To evaluate the presence of type I collagen, a primary antibody (C2456, Sigma), was used. Anti-mouse secondary antibody (BA-1000, Vector laboratories) was used in immunohistochemistry, followed by ABC staining, diaminobenzidine labelling, and counterstaining using hematoxylin. To evaluate the presence of Matrix-metalloproteinase 9, a primary antibody (ab38898, abcam) was used. Anti-rabbit secondary antibody (BA-2000, Vector laboratories) was used in immunohistochemistry, followed by ABC staining, diaminobenzidine labelling, and counterstaining using hematoxylin.

Yeung P., Sin H.S., Chan S., Chan G.C, & Chan B.P. (2015). Microencapsulation of Neuroblastoma Cells and Mesenchymal Stromal Cells in Collagen Microspheres: A 3D Model for Cancer Cell Niche Study. PLoS ONE, 10(12), e0144139.

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Immunohistochemical Analysis of CD4+ T Cells and Collagen I

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Frozen LN sections were fixed in Prefer for 15 minutes after equilibrating to room temperature. After rinsing in ethanol, slides were placed in distilled water then in tris‐buffered saline (TBS)+Tween 20. Staining was performed for CD4⁺ T cells (Abcam clone BC/1F6; 1:100 dilution) and collagen type I (COL1; Sigma‐Aldrich C2456; 1:500 dilution) in TBS+Tween and incubated over night at room temperature. Slides were rinsed in TBS+Tween and golden bridge‐ AP‐ polymer P1 rabbit was incubated for 30 minutes at room temperature. Slides were rinsed with TBS+tween then revealed using Fast Red chromogen for 10 minutes.

Rosen E.P., Deleage C., White N., Sykes C., Brands C., Adamson L., Luciw P., Estes J.D, & Kashuba A.D. (2022). Antiretroviral drug exposure in lymph nodes is heterogeneous and drug dependent. Journal of the International AIDS Society, 25(4), e25895.

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Evaluating TRICOL Decellularization and ECM

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Indirect immunofluorescence was performed on 5–6 µm-thick cryosections of unfixed NBPs, NPPs, DBPs and DPPs to evaluate the effectiveness of TRICOL decellularization and analyze extracellular matrix (ECM) composition. The primary antibodies used were collagen I (1:100, C2456; Sigma), elastin (1:100, E4013; Sigma), collagen IV (1:100, ab6586; Abcam, Cambridge, UK), laminin (1:500, Z0097; Dako Cytomation, Glostrup, Denmark), and heparan sulfate (1:100, MAB1948P; Millipore, Darmstadt, Germany). Controls were incubated with 1% (w/v) bovine serum albumin instead of the primary antibodies. Rhodamine-conjugated, anti-mouse, anti-rat and anti-rabbit IgGs (1:100, Millipore) were applied as secondary antibodies to reveal primary antibody binding. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired with an epifluorescence microscope Leica AF6000, connected to Leica DC300 digital camera and equipped with LAS AF Software (Leica Micro-systems, Wetzlar, Germany).

Zouhair S., Dal Sasso E., Tuladhar S.R., Fidalgo C., Vedovelli L., Filippi A., Borile G., Bagno A., Marchesan M., De Rossi G., Gregori D., Wolkers W.F., Romanato F., Korossis S., Gerosa G, & Iop L. (2020). A Comprehensive Comparison of Bovine and Porcine Decellularized Pericardia: New Insights for Surgical Applications. Biomolecules, 10(3), 371.

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Fibroblast Extracellular Matrix Production

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Normal human dermal fibroblasts
(NHDFs) from juvenile foreskin (j-NHDFs, Promocell C-12300) were plated
on 96-well plates at 6000 cells per well in fibroblast basal medium
(FBM, Lonza CC-3130) containing 2% fetal bovine serum (FBS) and cultured
under 5% CO₂ at 37°C. After 24 h, cells were switched
to FBM containing 0.5% FBS, containing 25 mg/mL Ficoll 400 and 100
μM l-ascorbic acid 2-phosphate (Sigma A8960). Small-molecule
treatments were applied at the same time as Ficoll 400.
Three inhibitors of proprotein convertase were p-guanidinomethyl-phenylacetyl-Arg-Val-Arg-4-amidinobenzylamide
(PCi, Calbiochem 537076), dec-RVKR-cmk (FiI, Tocris Bioscience 3501),
and hexa-d-arginine amide (FiII, Tocris Bioscience 4711).
After 3 days of biophysical crowding, cells were fixed in ice-cold
MeOH for 10 min at 4°C, blocked in 3% BSA for 30 min, and incubated
in 1/500 mouse monoclonal anticollagen type 1 (C2456, Sigma), 1/500
rabbit polyclonal antifibronectin (F3648, Sigma), or 1/100 rabbit
polyclonal anti-TGN46 (13573-1-AP, Proteintech) overnight. Subsequently,
goat antimouse IgG-Alexa488 and goat antirabbit IgG-Alexa647 (A-11001
and A-21245, respectively, Life Technologies) were used at 1/400 and
counterstained with Hoechst for 1.5 h. Signals of collagen type I
and fibronectin were imaged on the Operetta System (PerkinElmer).

Wang E.Y., Rafatian N., Zhao Y., Lee A., Lai B.F., Lu R.X., Jekic D., Davenport Huyer L., Knee-Walden E.J., Bhattacharya S., Backx P.H, & Radisic M. (2019). Biowire Model of Interstitial and Focal Cardiac Fibrosis. ACS Central Science, 5(7), 1146-1158.

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Immunostaining of Muscle Tissues

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Additionally, sections containing muscles were immunostained in batched sets by the same individual for alpha smooth muscle actin (αSMA, A2547, Sigma-Aldrich, Burlington, MA, USA, dilution 1:500) or collagen type I (C2456, Sigma-Aldrich, 1:500 dilution in PBS), using previously described methods [15 (link)]. DAPI was used as a nuclear stain (62246, Thermo Fisher Scientific, Rockford, IL, USA; diluted 1: 2000 with PBS for 15 min) before coverslipping with 80% glycerol in PBS. Additional sections containing median nerve branches were similarly immunostained for collagen type 1.

Lambi A.G., DeSante R.J., Patel P.R., Hilliard B.A., Popoff S.N, & Barbe M.F. (2023). Blocking CCN2 Reduces Established Palmar Neuromuscular Fibrosis and Improves Function Following Repetitive Overuse Injury. International Journal of Molecular Sciences, 24(18), 13866.

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Immunofluorescence Staining of Tissue Sections

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Cross-sections (7 μm cryosections from formalin-fixed samples) were washed in PBS (3 × 5 min), permeabilized in 0.5% Triton X-100 (30 min), and blocked for non-specific binding in 5% goat serum containing 1% BSA (30 min). The sections were then incubated with primary antihuman polyclonal antibodies against Ki67 (rabbit IgG, 1:200, Thermoscientific, RB-1510-P0), CD45 (mouse IgG1, 1:1000, Abcam, ab33533), vimentin (mouse IgM, 1:2000, Abcam, ab20346), αSMA (rabbit IgG, 1:600, Abcam, ab5694), or collagen I (mouse IgG1, 1:200, Sigma, c2456), in 10× diluted block solution (overnight at 4 °C). After washing in PBS (3 × 5 min), the sections were incubated with 1:500 secondary goat antibodies labeled with Alexa-488 conjugate (antimouse IgG1 (for CD45, Molecular Probes, A21121) or antirabbit IgG (for αSMA, Molecular Probes, A11008)) or Alexa-647 conjugate (antimouse IgM (for vimentin, Jackson Immunoresearch, 115-605-075) or antimouse IgG1 (for collagen I and Ki67, Molecular Probes, A21240)), in 10× diluted block solution (60 min). Nuclei were stained with 4′,6-diamidino-2-phenylindole (1:500, Sigma). The stained sections were mounted in mowiol (Sigma, 81381) and visualized with an inverted epifluorescent microscope (Zeiss Axiovert 200M, ×20/0.5 Plan-Neofluar lens, three sections/sample).

van Haaften E.E., Quicken S., Huberts W., Bouten C.V, & Kurniawan N.A. (2021). Computationally guided in-vitro vascular growth model reveals causal link between flow oscillations and disorganized neotissue. Communications Biology, 4, 546.

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