Heparin beads (Sigma) were soaked in 1 mg/ml of recombinant human FGF4 (R&D Systems) for 30 min on ice. FGF4 beads were grafted into the right wings of chick embryos at E4.5 and embryos were harvested 24 or 48 hours after grafting. Grafted right and contralateral left limbs were processed for in situ hybridization to sections.
Heparin beads
Manufactured by Merck Group
Sourced in United States
Heparin beads are a type of lab equipment used for various applications. They consist of heparin, a naturally occurring anticoagulant, immobilized on a solid support. Heparin beads can be used for the purification and separation of proteins and other biomolecules. Their core function is to provide a platform for affinity-based chromatography and binding studies.
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Lab products found in correlation
U0126, by Cell Signaling Technology (2 mentions)
Recombinant human fgf4, by R&D Systems (1 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
Fibroblast growth factor 2 (fgf2), by R&D Systems (1 mentions)
Collagenase, by Thermo Fisher Scientific (1 mentions)
Fgf10, by R&D Systems (1 mentions)
Type 2, by Thermo Fisher Scientific (1 mentions)
Affi gel blue beads, by Bio-Rad (1 mentions)
Fgf10, by Merck Group (1 mentions)
Su5402, by Merck Group (1 mentions)
Ag1 x2 beads, by Bio-Rad (1 mentions)
Picropodophyllotoxin ppp, by Bio-Techne (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Gradient tris glycine precast gel, by Bio-Rad (1 mentions)
Phosphor vegfr2, by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Merck Group (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Vegfr2, by Cell Signaling Technology (1 mentions)
Cleaved parp, by BD (1 mentions)
Tgfb1, by Thermo Fisher Scientific (1 mentions)
9 protocols using heparin beads
1
FGF4 Bead Grafting in Chick Limb
2
Chick Embryo Development Protocols
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Fertilized hens’ eggs (Brown Bovan Gold; Henry Stewart and Company) were incubated at 38 °C to the desired stages, following the Hamburger and Hamilton system (Hamburger and Hamilton, 1951 (link)). Electroporation, whole-mount in situ hybridization and whole-mount immunostaining were performed using standard methods as previously described (Sheng et al., 2003 (link), Stern, 1993 , Streit and Stern, 2001 (link), Voiculescu et al., 2008 (link)). All DNA solutions for electroporation were used at 1.5 μg/µl. FGF8 (50 μg/ml) and Calreticulin (50 μg/ml) proteins were delivered on heparin beads (Sigma; prepared as described by Streit et al., 2000 (link)).
3
Chick Embryo Retinectomy and FGF2/Wnt Modulation
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Retinectomies were performed in eyes of chick embryos at E4 (HH stage 22–24) using fine forceps as previously described [8] (link), [9] (link). For FGF2 treated eyes, heparin beads (Sigma, St. Louis, MO, USA) containing FGF2 (R&D System, Minneapolis, MN, USA) were prepared and added to the eyes as described in [9] (link). For the inhibitory experiments, 5 ul of 2 mM Wnt/β-catenin signaling inhibitor XAV939 (Reagents Direct, Encinitas, CA, USA) or PBS was injected into the vitreous chamber 30 minutes post-retinectomy. Embryos were collected 1, 3 or 7 days post-retinectomy (d PR) and processed as described below for histology and immunohistochemistry.
4
Mandibular Explant Culture Protocols
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Mandibular slice cultures were performed as previously described (Wells et al., 2013 (link); Li et al., 2016 (link)). For the bead experiment, two types of beads were used to help distinguish between the control and treated conditions. For the Fgf10-treated explants, heparin beads (Sigma, 100-200 mesh) were incubated overnight at 4°C with 100 μg/ml Fgf10 (R&D Systems). For the control, Affi-Gel blue beads (Bio-Rad,153-7302) were treated with 0.5% BSA. For inhibiting Fgf receptor signalling or the Erk pathway, explant cultures were treated with 2.5 μM SU5402 (Merck) or 5 μM U0126 (Cell Signaling Technology), respectively, made up in DMSO. Control cultures were treated with equivalent concentrations of DMSO (0.25% DMSO for the SU5402 and 0.5% DMSO for the U0126 experiment). For the collagenase treatment, whole E12.5 submandibular glands were dissected and treated for 2 days with 1 μg/ml collagenase, Type II (Thermo Fisher Scientific) and HBSS-treated glands were used as a control. Spooner ratios were calculated as the number of buds at the end of culture divided by the number of buds at the start of culture.
5
Grafting Heparin and Inhibitor Beads
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Heparin beads (Sigma H-5263) were soaked in recombinant human IGF-I or IGF-II (Peprotech) at 1mg/ml in phosphate buffered saline (PBS) with 0.1% Bovine Serum Albumin (BSA). AG 1-X2 beads (BioRad) were incubated in Picropodophyllotoxin (PPP, Tocris Bioscience), U0126 (Cell Signaling), LY 294002 (Calbiochem) or SU5402 (Calbiochem), all reconstituted in DMSO at 10mM. Beads were incubated for at least one hour in the dark before being washed briefly in 2% phenol red and rinsed in PBS before grafting. Beads were grafted into limb buds with a sharpened tungsten needle, resealed with sellotape and reincubated for 18-48h as described previously [31 (link)].
6
Heparin Bead Implantation for FGF Signaling
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The method described in [9 (link)] was followed. Briefly, Heparin beads (Sigma H-5263) were washed three times in PBS before soaking for 1 hour at room temperature in recombinant FGF (R&D Systems) at the following concentrations: FGF2 (400μg/ml) and FGF4 (50μg/ml). After washing twice in PBS, beads were implanted adjacent to forelimb or flank level somites of HH16 embryos. Control beads were soaked in PBS. Embryos were allowed to develop for 24 hours and were then analysed by whole mount in situ hybridisation.
7
Protein Expression Analysis in Retinal Lysates
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Protein expression in retina lysate was analyzed as described previously [23] (link). For VEGF, retinal lysates were subjected to heparin beads (Sigma) as described before [27] (link), [28] (link). The beads were pelleted at 5000 × g for 1 min, washed in 400 mM NaCl and 20 mM Tris and loaded onto a 4-20% gradient Trisglycine pre-cast gel (BioRad). The primary antibodies were purchased as follow: VEGF (Rabbit polyclonal, EMD-Millipore), phosphor-VEGFR2, VEGFR2, phospho-Akt, Akt, phospho-ASK-1, ASK-1, cleaved caspase-3 (Rabbit polyclonal, Cell Signaling Tech, Danvers, MA), total Trx (Mouse monoclonal, Santa Cruz, Dallas, TX), and TXNIP (Rabbit polyclonal, Invitrogen, Grand Island, NY), cleaved PARP (BD Bioscience Pharmingen, San Diego, CA). Primary antibodies were detected using a horseradish peroxidase-conjugated antibody and enhanced chemiluminescence (GE Healthcare, Piscataway NJ).The films were scanned, and band intensity was quantified using densitometry software (Alpha Innotech Fluorchem, Santa Clara, CA).
8
Embryonic Tongue Recombination and FGF10
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E12.5 embryonic tongues were dissected and incubated in 2.2 U/ml Dispase II for 50 min at 37°C and washed in basal medium containing 10% FBS. Tongue epithelium and mesenchyme were gently separated in cold medium. The epithelium was placed 180° rotated anterior-posterior. Heparin beads (100–200 mesh; Sigma Aldrich, United States) soaked with 100 μg/ml FGF10 protein (6224-FG-02S; R&D Systems, United States) were implanted between the recombined CVP epithelium and non-CVP mesenchyme at E12.5 and cultured for 48 h as in both groups. The CVP epithelium and underlying mesenchyme combined at original as control. Specimens from 4 embryos were examined for each group.
9
Hindlimb Tendon Induction Assay
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Fertilized White Leghorn chicken eggs were obtained from Alpes, Puebla, Mexico. They were incubated at 38°C, and the embryonic chick hindlimbs at stage 28HH (Hamburger & Hamilton, 1951) were used for all experiments. For treatments, heparin beads (Sigma-Aldrich, St. Louis, MO, USA) were soaked in 1 mg/ml of human recombinant WNT3A or 1 mg/ml DKK (Preprotech, Mexico City, Mexico). DKK1 is a high affinity antagonistic for the WNT co-receptor LRP6. Affi-gel agarose beads (Bio-Rad Laboratories Inc., USA) were soaked in 200 ng/ml TGFb1 (Peprotech). AG1-X2 acetate beads (Bio-Rad Laboratories Inc., USA) were soaked in 50mM SB431542 (ALK inhibitor) or 20mM SIS3 (SMAD inhibitor). Protein-or chemical-soaked beads were implanted on the dorsal side of the hindlimb at the level of the third metatarsal. All handlings were performed in the presumptive tenogenic area limited by the proximal phalange three and the distal border of the Extensor Digitorum Communis (EDC) tendon (Pryce et al., 2009) . For all experiments, the right hindlimb was exposed for surgical manipulation, and left hindlimb was used as a control. After treatment, the chick embryos were returned to the incubator and collected after 2h or 4h for gene expression analysis by in situ hybridization.
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