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Immunoblotting analysis of formalin‐fixed and paraffin‐embedded malignant tumor samples (1048.16)
Author(s) -
Celes Mara,
Soave Danilo,
Freitas Ana Caroline,
Ramos Simone,
RibeiroSilva Alfredo
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1048.16
Subject(s) - antigen retrieval , protein purification , usable , pathology , chemistry , lysis buffer , bradford protein assay , microbiology and biotechnology , lysis , immunohistochemistry , biology , chromatography , medicine , computer science , world wide web
Formaldehyde‐fixed, paraffin‐embedded (FFPE) tissue remains the gold standard technique used for preservation and storage of tissue specimens for pathologic analysis. FFPE‐tissues represent invaluable resources for conducting retrospective disease investigations. Several studies have been undertaken to obtain usable quantities of proteins from FFPE‐samples. Objective: This study was performed to describe and analyze the potential usefulness of protein extraction protocol from paraffined samples of tumor lesions (salivary gland and lung). Methods: Paraffin was removed from tissue samples 15 micrometers thick sections with an organic solvent, followed by protein extraction by a modified RIPA lysis buffer and solubilization in 5% SDS. The resultant protein extract was resolved by SDS‐PAGE and submitted to immunoblot analyses using the antibodies to CD31 and GAPDH. Results: SDS‐PAGE and immunoblot analysis presented usable quantities of protein. However, FFPE‐tissues preserved for 10 years presented less usable proteins than FFPE‐samples preserved for 2 years. In addition, protein extraction from salivary glands tissue provided more usable protein than the lung tumor tissues. Conclusion: In summary, our investigation presented evidence that the evaluation of protein extraction in FFPE‐samples can be used to provide improved information about tumors protein expression. We also believe that some factors such as tissue density, kind and time preservation of FFPE‐samples may influence the quality and quantity of usable proteins. Grant Funding Source : Supported by FAPESP and FAPEG