For in vitro studies, VX-509 (Cat# B5929, APE × BIO, United States) was dissolved in dimethyl sulfoxide (DMSO, MP Biomedicals, United States) at a stock solution concentration of 10 mmol/L. For in vivo studies, VX-509 was dissolved in the mixture containing DMSO and 5% sodium carboxymethylcellulose (CMC). Recombinant Nodal recombinant homolog was purchased from Zeye company (Shanghai, China). Nodal recombinant protein was prepared as stock solutions in phosphate-buffered saline (PBS) prior to diluting in cell culture medium. For antibodies, Oct-4 (Cat# 381335, ZEN-BIOSCIENCE, Chengdu, China), Nanog (Cat# 381167) and SOX2 (Cat# 864316) were purchased from ZEN-BIOSCIENCE; CD133 (Cat# ab216323), CD44 (Cat# ab189524) and Nodal (Cat# ab55676) were purchased from Abcam; β-actin (Cat# 20536-1-AP) and GAPDH (Cat# 10494-1-AP) were purchased from Proteintech; E-cadherin (Cat# AF6759), N-cadherin (Cat# AF5237), vimentin (Cat# AF1975), JAK3 (Cat# AF7314), p-Smad2/3 (Cat# AF5920), goat anti-rabbit immunoglobulin (Ig)G antibody (Cat# A0208) and goat anti-mouse IgG antibody (Cat# A0216) were purchased from Beyotime.
Nodal
Manufactured by Abcam
Sourced in China, United States
NODAL is a recombinant protein produced in E. coli. It is a member of the TGF-beta superfamily and plays a key role in the regulation of embryonic development and stem cell differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse igg antibody, by Beyotime (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Beyotime (1 mentions)
Vimentin, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Gapdh, by Proteintech (1 mentions)
Dimethyl sulfoxide (dmso), by MP Biomedicals (1 mentions)
Fibronectin, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Affinity Biosciences (1 mentions)
E cadherin, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Affinity Biosciences (1 mentions)
Vimentin, by Affinity Biosciences (1 mentions)
Jam a, by Thermo Fisher Scientific (1 mentions)
Image pro plus 6.0 image, by Media Cybernetics (1 mentions)
Vimentin, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
4 protocols using nodal
1
Pharmacological Evaluation of VX-509 and Nodal Protein
2
Immunohistochemical and Immunofluorescent Analyses
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Immunohistochemical analyses were performed on 5μm paraffin sections with an indirect immunoperoxidase method by antibodies against JAM-A (Life Technologies, 1:100 dilution), E-Cadherin, Vimentin, Fibronectin (Affinity biosciences, Piscataway, NJ, USA, 1:200 dilution) and NODAL (Abcam, 1:200 dilution). Immunofluorescent assays were performed on acetone-fixed cells with rabbit anti-human-E-Cadherin, Vimentin and Fibronectin as primary antibodies, and Alexa Fluor-conjugated donkey anti-mouse-IgG and anti-rabbit-IgG antibodies (Invitrogen, Carlsbad, CA, USA) as secondary antibodies. Nuclei were stained by DAPI.
The protein expression levels were scored based on staining intensity (SI) and distribution using the immunoreactive score (IRS). Briefly, IRS = SI × PA (positive area). The intensity of immunohistochemical and immunofluorescence staining was measured by the Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Silver Spring, USA)29 (link).
The protein expression levels were scored based on staining intensity (SI) and distribution using the immunoreactive score (IRS). Briefly, IRS = SI × PA (positive area). The intensity of immunohistochemical and immunofluorescence staining was measured by the Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc., Silver Spring, USA)29 (link).
3
Immunohistochemistry of Colon Cancer
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Colon cancer tissues and reciprocally adjacent noncancerous colon tissues were obtained from Renji Hospital, Shanghai Jiao Tong University School of Medicine. To prepare sections, colon cancer tissues and the matched noncancerous colon tissues were fixed in 4% PFA for 3 hours, dehydrated through a series of graded alcohols, embedded in paraffin at 60°C overnight, and sectioned at 5 μm thickness.
Immunohistochemistry was performed in triplicate in human colon cancer tissues and noncancerous colon tissues using antibodies against NODAL (Abcam), ALK-4 (Santa Cruz), CD24 (Abcam), and CD44 (Abcam) at a dilution of 1 : 200 in these tissues according to the procedure as previously described [23 (link)].
Immunohistochemistry was performed in triplicate in human colon cancer tissues and noncancerous colon tissues using antibodies against NODAL (Abcam), ALK-4 (Santa Cruz), CD24 (Abcam), and CD44 (Abcam) at a dilution of 1 : 200 in these tissues according to the procedure as previously described [23 (link)].
4
Nodal Signaling Regulation in Colorectal Cancer
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HCT116 cells, CD24-negative HCT116 cells, and CD24-positive HCT116 cells cultured without NODAL or with NODAL were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology). After 30 min lysis on ice, cell lysates were cleared by centrifugation at 12,000 g, and the concentration of protein was measured by BCA kit (Dingguo Company, China). Fifty micrograms of cell lysate from each sample was used for SDS-PAGE (Bio-Rad Laboratories, Richmond, CA), and Western blots were performed in triplicate according to the protocol as described previously [23 (link)]. The primary antibody included NODAL (Abcam), phos-Smad2 and phos-Smad3 (Cell Signaling Inc.), and ACTB (Abcam). After extensive washes in PBS, the blots were detected by chemiluminescence (Chemi-Doc XRS, Bio-Rad, Hercules, CA).
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