ABSTRACT
WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.
Subject(s)
Antibodies/immunology , Wnt-5a Protein/immunology , Biomarkers, Tumor , Blotting, Western , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Reproducibility of Results , Staining and Labeling , Wnt-5a Protein/chemistryABSTRACT
Recently, the fingerprint approach using chromatography has become one of the most effective tools for quality assessment of herbal medicines and food supplements: due to the complexity of the chromatographic fingerprint and the irreproducibility of chromatographic instruments and experimental conditions, chemometric approach is employed to deal with the chromatographic fingerprint. The study was aimed at developing new analytical methods for the multivariate phytochemical fingerprinting of bioactive compounds in eight tree-species bud-preparations, commonly used in phytotherapy. Methods was used to identify and quantify the main bioactive compounds (polyphenols, organic acids and vitamins), and obtain a specific botanical profile in order to assess the contribution of each single bioactive class to the total bud preparation phytocomplex. A chemometric approach was used to distinguish among different genotypes assuring the identity, safety and quality of the botanical raw materials. The established protocol was simple, sensitive and reliable and it could be used for the evaluation and quality control of bud-extracts and natural food supplements: the proposed method was successfully applied to the characterization of commercial bud-preparations, demonstrating to be an effective tool for the fingerprinting of this plant material. The new approach developed in this study represents a good alternative for improving the classification results of herbal materials with complex chromatograms. It should be necessary to develop a "multivariate chromatographic fingerprint", in order to differentiate the herbal preparations according to their genotype, avoiding substitutions, changes or adulterations with other species or synthetic drugs.
ABSTRACT
Amelanchier canadensis (L.) Medik., commonly called serviceberry, is a potential functional food that is also used for its medicinal purposes. This work evaluated the potential of a cultivated serviceberry species as a functional food by characterising its physicochemical characteristics, antioxidant capacity, vitamin C, phenolics and other phytochemicals selected as health-promoting biomarkers, using high-performance liquid chromatography. The most important compound class identified was polyphenols (62.10%), followed by organic acids (22.63%), monoterpenes (7.95%), and vitamins (7.32%). Results showed that serviceberry fruits could be good sources of phenolic constituents, as catechins (343.46 ± 29.46 mg/100 gFW), anthocyanins (220.66 ± 17.43 mg/100 gFW), and tannins (209.29 ± 7.81 mg/100 gFW) (FW = fresh weight). These results highlight the potential role of A. canadensis fruits as a functional food. Further studies are needed to identify several genotypes for breeding to get suitable cultivars for fresh consumption and processing.
