Specimens were decalcified and embedded in paraffin, and each specimen was cut into 5 μm sections. Hematoxylin and eosin staining (H&E) and Goldner's trichrome were performed to visualize bone, cartilage, and blood vessel formation in the bone defect area. Immunohistochemical staining was used to identify CCR7 (1:50, Abcam), CD206 (1:50, Santa Cruz Biotechnology), VEGF (1:200, Abcam), PDGF-bb (1:50, Abcam), CD31 (1:200, Santa Cruz Biotechnology) at the defect area. Images were observed under a light microscopy (ZEISS, Germany).
Pdgf bb
Manufactured by Abcam
Sourced in United Kingdom, United States
PDGF-BB is a recombinant human platelet-derived growth factor BB homodimer. It is a member of the platelet-derived growth factor family and plays a role in cell growth and division.
Automatically generated - may contain errors
Lab products found in correlation
Vegfa, by Abcam (2 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Light microscopy, by Zeiss (1 mentions)
Cd206, by Santa Cruz Biotechnology (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Endomucin, by Santa Cruz Biotechnology (1 mentions)
11β hsd1, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Pdgfrβ, by Abcam (1 mentions)
Vegfr2, by Abcam (1 mentions)
Fgf 2, by Novus Biologicals (1 mentions)
Fgfr1, by Novus Biologicals (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Calponin, by Abcam (1 mentions)
Nucleolin, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Sm22a, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
8 protocols using pdgf bb
1
Histological Evaluation of Bone Regeneration
2
Femoral Tissue Immunofluorescence Analysis
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The expression of TRAP (Santa Cruz), 11β-HSD1 (Proteintech), Endomucin (Santa Cruz), CD31 (R&D), PDGF-BB (Abcam) and YAP (Abcam) in femoral tissues was analyzed by immunofluorescence. The staining method was similar to that of our previous published article47 (link). Finally, the positively stained cells were scanned using fluorescence microscope (Zeiss Axiovert 200; Carl Zeiss Inc., Thornwood, NY, USA). Quantitative analysis was done with the aid of ImageJ (National Institutes of Health, NIH)48 (link).
3
Angiogenic Growth Factors in Rabbit Aorta
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Proteins were extracted from rabbit aortas. Tissues were lysed in lysis buffer (100 mM Tris-Cl, pH 6.8, 4%(m/v) SDS, 20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM PMSF, and 1 g/ml aprotinin) and proteins were transferred to PVDF membranes (0.45 mm, Millipore), which were then incubated overnight at 4°C with primary antibodies for VEGF-A (1:200, Abcam), VEGFR-2 (1:200, Abcam), FGF-2 (1:500, Novus), FGFR-1 (1:500, Novus), PDGF-BB (1:200, Abcam), PDGFR-β (1:200, Abcam), and β-actin (1:1000, Cell Signaling Technology). Protein bands on the membrane were visualized using chemiluminescence (Millipore) and were quantified by densitometry.
4
Protein Expression Analysis of VSMC Cells
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Following various treatments, VSMCs cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (Shanghai Biyuntian Biotechnology, Ltd.). The protein concentration was determined using BCA assay. Whole‐cell lysates were subjected to SDS‐PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. The blots were incubated with respective primary antibodies against nucleolin (rabbit polyclonal antibody, Sigma), α‐SM‐actin (mouse monoclonal antibody, Boster Biotech), SM22a (rabbit polyclonal antibody, Abcam), calponin (mouse monoclonal antibody, Abcam), OPN, EGF, PDGF‐BB (rabbit polyclonal antibody, Abcam) and β‐actin (mouse monoclonal antibody, Abcam) at 25°C for 2 hours. Subsequently, the blots were incubated with peroxidase‐conjugated secondary antibodies at 25°C for 1 hours. Immunoreactive bands were visualized utilizing enhanced chemiluminescence detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions, and the densitometry analysis was performed by scion image software.
5
Quantifying Growth Factor Release from A-PRF
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To measure growth factor release, both A-PRF preparations were transferred into a 6-well plate. One milliliter of DMEM (Gibco, Life Technologies Corporation, New York, NY, USA) was added to each sample. The plate was then placed in a 5% CO2 incubator at 37 °C to allow for growth factors to be released into the culture media during the three-day period of the study. At 1 h, 24 h, and 72 h, 1 mL of the culture media was collected, kept at −20 °C, and replaced with 1 mL of additional fresh culture media. The collected samples at each time point were analyzed for canine TGF-β1, VEGFA, and PDGF-BB using commercial ELISA kits, including TGF-β1 (MyBioSource, Inc., San Diego, CA, USA), VEGFA (Abcam, Cambridge, UK), and PDGF-BB (Abcam, Cambridge, UK), respectively, according to the manufacturers’ instructions. Optical density was assessed using a microplate reader at 450 nm. The absorbance measured at 630 nm was subtracted from that measured at 450 nm for an optical density correction. The measurement was performed in duplicate.
6
Quantitative Western Blot Analysis
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Western blotting was performed, and results were analyzed as described previously [23 (link)]. The following primary antibodies were used at the indicated dilutions: GAPDH (1:3000, mouse, Santa Cruz), PDGF-BB, Col I, semaphorin7A, CXCL12, and α-SMA (all at 1:2000, Abcam, Cambridge, MA). The membranes were then incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody, and the target signals were detected using the ECL Plus Western Blot Kit (Amersham Pharmacia Biotech, NJ).
7
Modulation of Inflammatory Cytokines in Hepatic Stellate Cells
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The levels of IL-1β, IL-6 and TNF-α in serum were measured by ELISA detection kits (R&D, USA) according to the operation manual. The mRNA levels in the liver were detected by RT-PCR. Results were from triplicate experiments.
Cell culture and siRNA-β-klotho transfection Rat hepatic stellate cell line was purchased from American Type Culture Collection. The cells were grown in DMEM + 10% foetal bovine serum, 1% penicillin/streptomycin at humidity atmosphere of 5% CO 2 at 37℃. PDGF-BB (Abcam, USA) was used to activate HSCs following previous study. The cells were divided into four groups in the logarithmic phase of growth: the PBS group; The PDGF-BB group (20ng/ml; The PDGF-BB + FGF21 (0.1 µM); The PDGF-BB + FGF21 (1.0 µM), namely C, P, P + FL and P + FH. The β-klotho mRNA was speci cally knocked down by using commercially available siRNA oligonucleotides. The sequences of the siRNA were designed by Sangon Biotech (Shang hai, China). The negative siRNA was used as control.
Cell culture and siRNA-β-klotho transfection Rat hepatic stellate cell line was purchased from American Type Culture Collection. The cells were grown in DMEM + 10% foetal bovine serum, 1% penicillin/streptomycin at humidity atmosphere of 5% CO 2 at 37℃. PDGF-BB (Abcam, USA) was used to activate HSCs following previous study. The cells were divided into four groups in the logarithmic phase of growth: the PBS group; The PDGF-BB group (20ng/ml; The PDGF-BB + FGF21 (0.1 µM); The PDGF-BB + FGF21 (1.0 µM), namely C, P, P + FL and P + FH. The β-klotho mRNA was speci cally knocked down by using commercially available siRNA oligonucleotides. The sequences of the siRNA were designed by Sangon Biotech (Shang hai, China). The negative siRNA was used as control.
8
Bone Fracture Microfluidic Chemoattraction
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In order to recreate the local conditions that occur in the environment of a bone fracture or injury, we induce in the microfluidic device a chemical gradient using PDGF-BB as chemoattractant. The concentrations of the recombinant human PDGF-BB (Invitrogen) isoform used in the experiments for the application of the chemical gradient with human osteoblasts were 5ng/ml and 50ng/ml respectively. After 24h of incubation, PDGF-BB (Abcam) was added achieving a gradient across the gel by adding the growth factor (5ng/ml or 50ng/ml) containing culture medium to only one channel, while new medium alone was added to the other channel. The chemical gradient was established by a diffusive process across the hydrogel (Moreno-Arotzena et al, 2014; Moreno-Arotzena et al, 2015; Del Amo et al, 2016) .
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