The level of folate in serum was measured by FA/VB9 Elisa kit (E-EL-0009c, Elabscience) according to manufacturer’s instructions. For western blotting, Tissues or cells were collected and lysed in universal lysis/immunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 25 mM NaFl, 25 mM β-glycerophosphate pH 7.5, 0.1 mM sodium orthovanadate, 0.1 mM PMSF, 5 μg of leupeptin per ml, 0.2% Triton X-100, 0.5% Nonidet P-40). The concentrations of the total lysate protein were measured by a standard Bradford assay (Bio-Rad, San Diego, CA). The protein was electrophoresed by S.D.S-PAGE and transferred to nitrocellulose membrane (PierceChemical) and probed with primary antibodies. The primary antibodies were as follows: p-AMPKα (cell signal 2535L, 1:1000), p-ACC (cell signal 3661L, 1:1000), AMPKα (cell signal 5832, 1:1000), ACC (cell signal 3662L, 1:1000), β-Actin (Santa Cruz sc-1615, 1:1000), MBP (Covance SMI-94R, 1:1000), PDGFRα (Santa Cruz sc-338, 1:500). Appropriate secondary antibodies and ECL were performed to visualize the protein signaling.
Sc 338
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-338 is a laboratory equipment designed for general use in scientific research. It is a multi-functional device capable of performing various tasks. The core function of Sc-338 is to provide a controlled environment for conducting experiments and analyses. No further details on the intended use or interpretation of this product are available.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
γ tubulin, by Merck Group (1 mentions)
Ab187284, by Abcam (1 mentions)
Ab150075, by Abcam (1 mentions)
Facscanto 2, by BD (1 mentions)
Ab28364, by R&D Systems (1 mentions)
A7811, by Merck Group (1 mentions)
Alexa fluor 647 secondary antibody, by Thermo Fisher Scientific (1 mentions)
A2547, by Abcam (1 mentions)
Sc 432, by Santa Cruz Biotechnology (1 mentions)
Sc 8035, by Santa Cruz Biotechnology (1 mentions)
Interferin sirna transfection reagent, by Polyplus Transfection (1 mentions)
Abe419, by Merck Group (1 mentions)
Bond max, by Leica camera (1 mentions)
Ab10135, by Abcam (1 mentions)
Ab9361, by Abcam (1 mentions)
A21432, by Thermo Fisher Scientific (1 mentions)
A11055, by Thermo Fisher Scientific (1 mentions)
A21206, by Jackson ImmunoResearch (1 mentions)
11 protocols using sc 338
1
Folate Quantification and Protein Analysis
2
Immunohistochemistry of PDGFRα in Tissue
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For immunohistochemistry studies tissue was fixed in 4% paraformaldehyde overnight. The following day, the tissue was transfer to 20% sucrose in phosphate buffer and incubate overnight, and OCT embedding tissues were frozen in liquid nitrogen and store at −80 °C until use. Cryosections (8 µm thickness) were cut by cryostat. The sections were incubated in 0.01 M PBS containing 0.3% Triton-X (PBS-T) and treated with 2% bovine serum albumin (BSA) for 60 min at room temperature (RT). After washing with 0.01 M PBS-T, the sections were incubated with primary antibodies at 4 °C overnight, followed by secondary staining. Primary antibodies used were anti PDGFRα (1:200; SC-338, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies used Alexa Fluor 647 conjugated IgG (1:800; 111-606-003, Jackson immunoResearch, West Grove, PA, USA). Nuclei were stained using 4’,6-diamidino-2-phenylindole (DAPI) (1:1000; D523, Dojindo, Kumamoto, Japan). Sections were observed with a confocal laser scanning microscopy (Nikon/A1; Nikon, Tokyo, Japan).
3
Neonatal Rat Brain Development
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Unless specified otherwise, all chemicals were obtained from Sigma-Aldrich. Antibodies were: acetylated α-tubulin (T7451, Sigma-Aldrich, and D20G3; Cell Signaling Technology), ADP-ribosylation factor-like 13B (Arl-13B, NeuroMab clone N295B/66), Glutamylated tubulin (AB3201, Chemicon), Myelin basic protein (MBP, Aves Labs), γ-tubulin (T6557, Sigma-Aldrich), Histone-3P (06–570, Millipore), BrdU (M0744, DAKO) and PDGF-Rα (SC-338, Santa Cruz Biotech).
This study was carried out strictly in accordance with animal use guidelines of the National Fund for Science and Technology (FONDECYT) and with the Guide for the Care and Use of Laboratory Animals issued by the Institute for Laboratory Animal Research of the National Research Council (USA; National Academies Press, 2011). All procedures were approved by the Ethics Committee of the Faculty of Sciences, the University of Chile (certificates issued on July 31st, 2012 and October 4th, 2013) and were reviewed by the Bioethics Committee of the National Fund for Science and Technology (FONDECYT). Animals were neonatal (P1) Sprague Dawley rats obtained from the central animal housing facilities at the Catholic University of Chile. Suffering of animals was kept to a minimum and no procedures inflicting pain were performed.
This study was carried out strictly in accordance with animal use guidelines of the National Fund for Science and Technology (FONDECYT) and with the Guide for the Care and Use of Laboratory Animals issued by the Institute for Laboratory Animal Research of the National Research Council (USA; National Academies Press, 2011). All procedures were approved by the Ethics Committee of the Faculty of Sciences, the University of Chile (certificates issued on July 31st, 2012 and October 4th, 2013) and were reviewed by the Bioethics Committee of the National Fund for Science and Technology (FONDECYT). Animals were neonatal (P1) Sprague Dawley rats obtained from the central animal housing facilities at the Catholic University of Chile. Suffering of animals was kept to a minimum and no procedures inflicting pain were performed.
4
Cardiac Tissue Cell Isolation and Culture
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TC content in the enriched cultures was assessed by double labeling with phycoerythrin-conjugated monoclonal anti-CD34 (ab187284; Abcam, Cambridge, MA, USA) and rabbit polyclonal anti-PDGFRα (sc-338; Santa Cruz Biotechnology, Dallas, TX, USA). The TCs were analyzed using a BD FACS CantoII cytometer (BD Biosciences, San Jose, CA, USA). Passage-2 cells were washed with D-PBS and harvested by trypsinization. The samples were stained with the above antibodies for 1 h at room temperature. Cells were then incubated with donkey anti-rabbit H&L-labeled secondary antibodies (ab150075; Abcam) for 30 min at room temperature. As a negative control, unstained cell aliquots were incubated with D-PBS under the same conditions. According to the results of the identification, the flow cytometry was adjusted to set the gate and CD34+/PDGFRα+ cells were sorted to purify the cardiac TCs. The sorted cells were collected in a collecting tube, which was prefilled with complete culture medium and 2% PS, and then centrifuged at 300 × g for 5 min. The cell density was adjusted to 1×105/ml with DMEM/F12 medium containing 10% FBS and 1% PS, following which the cells were seeded into sterile culture dishes and cultured in a humidified atmosphere of 5% CO2 at 37°C.
5
Immunohistochemical Staining of PDGFRa
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Immunohistochemical stain using mouse mono-clonal antibody against PDGFRa (SC-338, Santa Cruz, CA, 1:50 dilution) was performed as previously described on other studies [31 (link)]. The result was interpreted in a semi-quantitative manner. In cases of positive cells < 5% was considered as negative stain, and those > 5% was positive. In the positive cases, the scoring was performed according to the strength of the immunostaining as weak staining, moderate staining, and strong staining.
6
Immunofluorescence Staining of Cardiac Markers
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Sections were washed three times with PBS followed by antigen retrieval in 0.25% Triton for 10 min. Samples were blocked for 1 h in 10 % goat serum followed by incubation with primary antibodies for 2 h at room temperature (RT). Antibodies against α-sarcomeric Actinin (1/400, Sigma, A7811), α-smooth muscle actin (1/100, Sigma, A2547), CD31 (1/100, Abcam, ab28364), DDR2 (1/100, R&D, MAB25381), WT1 (1/100, Abcam, ab89901), and PDGFRα (1/100, Santa Cruz, sc338) were used. Alexa fluor 647 secondary antibodies were used (1:100, Invitrogen) for 1 h at RT. Co-localization of proteins with Rainbow-labeled clones was obtained through confocal z-stack analysis. WGA (1/500, Invitrogen) staining was performed for 1 h at RT. For co-localization purposes, blue pseudocolor was assigned to all clones analyzed in this manuscript.
7
Silencing PDGFRβ in Mesenchymal Stem Cells
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cMSCs were transfected with 30 nM mouse PDGFRβ (or control) small interfering RNA (siRNA) (Santa Cruz Biotechnology) using INTERFERin siRNA transfection reagent (Polyplus Transfection) according to the manufacturer’s instructions. After 96 hours of transfection, cMSCs were evaluated for PDGFRβ expression by immunoblotting using standard protocols as previously described.22 (link),23 (link) In other experiments, siRNA–transfected cMSCs were trypsinized, washed, and counted 24 hours’ post-transfection followed by the collagen gel contraction assay as described earlier. Primary antibodies used for immunoblotting were purchased from Santa Cruz Biotechnology, including against PDGFRα (sc-338), PDGFRβ (sc-432), alpha smooth muscle actin (α-SMA) (sc-58669), α-tubulin (sc-8035), vinculin (sc-736), and glyceraldehyde-3-phosphate dehydrogenase (sc-32249).
8
Immunohistochemistry for CCND1, CSPG4, PDGFRA, and H3 K27M
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Immunohistochemistry was performed using a Leica Bond-Max autostainer according to manufacturer’s protocol F. Cases were stained for CCND1 (1/40, CellMarque–241R), CSPG4 (1/100, Atlas–HPA002951) and PDGFRA (1/200, Santa Cruz–sc-338). Immunohistochemistry against H3 K27M (1:1500, Millipore – ABE419) was performed in the same manner. Positive controls for all antibodies were included (CCND1-Mantle cell lymphoma; CSPG4-Large intestine, PDGFRA-Gastrointestinal stromal tumour; H3 K27M-Diffuse midline glioma, H3 K27M-mutant). Staining for CCND1 and PDGFRA was undertaken at UCL Advanced Diagnostics.
9
Comprehensive Immunofluorescence Staining Protocol
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The following chemical reagent and antibodies were used for immunofluorescence: Alexa Fluor 633 hydrazide (Elastin) (A30634, Invitrogen), goat anti-TDTomato (1:200, MBS448092, MyBioSource.com ), rabbit anti-Connexin 43 (Cx43) (1:200, SC-9059, Santa Cruz Biotechnology), mouse anti-Smooth Muscle Actin (αSMA) (1:300, Clone 1A4, Dako), goat anti-TAGLN (1:200, ab10135, Abcam), chicken anti-β Galactosidase (1:200, ab9361, Abcam), FITC-conjugated mouse anti-CD34 (1:200, 11–0341-82, Invitrogen), mouse anti-LEF1 (1:100, SC-374522, Santa Cruz Biotechnology), rabbit anti-PDGFRA (1:100, sc-338, Santa Cruz Biotechnology), chicken anti-GFP (1:200, AB-2307313, Aveslab), rabbit anti-MEOX2 (1:200, NBP2–30647, Novus Biological). Alexa-488, 555, 594 or Alexa-647-conjugated donkey anti-goat, anti-rabbit, anti-mouse, anti-chicken secondary antibodies (all from Invitrogen; A11055, A21432, A21447, A21206, A21207, A31573, A21202, A31570, A21203, A31571, A78952), Alexa-488 or Alexa 594- conjugated donkey anti-chicken secondary antibodies (703–545-155, and 703–585-155 from Jackson ImmunoResearch).
10
Western Blot Protein Detection
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Proteins were separated by weight in 12% polyacrylamide-SDS-gels and transferred after to PVDF-membranes (Carl Roth) using a semi-dry transblot system (Carl Roth). Membranes were blocked with 5% skimmed milk powder (Heirler, Radolfzell, Germany) in tris-buffered saline with Tween TBST (0.05% Tween-20, 1 × TBS; 10 × TBS: 250 mM Tris/HCl pH 7.4, 1.5 M NaCl) (blocking solution) for 1 h. Membranes were incubated in primary antibody, dissolved in blocking solution at 4 °C overnight, followed by three washing steps in TBST. Next, a one-hour incubation with the secondary antibody, which was dissolved in blocking solution, and finally, three washing steps with TBST and one wash with 1 × TBS were carried out. Western blots were developed after incubation with the substrate solution (ECL Substrate, BioRad Lab. Inc., Hercules, CA, USA) for 5 min. Antibodies: LRP1 (1:10,000, ab92544, Lot: 6R259330-27, Abcam), MBP (1:1000, MCA409S, Lot: 161031A, BioRad), PDGFRα (1:10,000, sc-338, Lot: E2015, Santa Cruz), α-tubulin (1:10,000, T9026, Lot: 078M4796 V, Sigma), Goat α rabbit HRP (1:5000, 111-035-144, Lot: 132409, Jackson ImmunoResearch Laboratories Inc.), Goat α mouse HRP (1:10,000, 115-035-068, Lot: 132223, Jackson ImmunoResearch Laboratories Inc.), and Goat α rat (1:5000, 112-035-062, Lot: 90553, Jackson ImmunoResearch Laboratories Inc.).
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