A novel xylene-free deparaffinization method for the extraction of proteins from human derived formalin-fixed paraffin embedded (FFPE) archival tissue blocks

Protein detection methods in formalin-fixed paraffin embedded (FFPE) tissue blocks are widely used in research and clinical setting in order to diagnose or to confirm a diagnosis of various types of diseases. Therefore, multiple protein extraction methods from FFPE tissue sections have been developed in this regard. However, the yield and the quality of proteins extracted from FFPE tissues are significantly reduced in blocks stored for longer periods of time. Regardless the protein extraction method used, tissue sections must be first deparaffinized with xylene, and then washed in serial dilutions of ethanol in order to remove the toxic organic solvent “xylene” and rehydrate the tissue. The objective of this study was first to develop a method to deparaffinize FFPE blocks that excludes the use of toxic solvent “xylene”. Second minimize the time required to perform the extraction. Here we describe a method where: • The entire paraffin embedded blocks are deparaffinized and rehydrated using only hot distilled water as a substitute for both xylene and ethanol • The entire procedure takes about 15 min • Deparaffinized blocks are immediately homogenized in lysis buffer, and the obtained lysate analyzed by Western blot. With this new modified technique, we were able to successfully detect actin and AKT proteins in lysates from blocks embedded in paraffin for up to 9 years

Irritable bowel syndrome: prevalence, risk factors in an adult Lebanese population

Abstract Background Very few studies report on the prevalence of irritable bowel syndrome (IBS) and its correlates in the Middle East. This study investigated Irritable Bowel Syndrome (IBS) prevalence in a sample of Lebanese adult individuals and associated demographic and behavioral lifestyle factors. Methods This is an observational population-based study. The target population is working Lebanese adults, eighteen-to-sixty five years old. The sample was selected from a convenience population of bank employees in different geographical areas in Lebanon. The study participants completed an anonymous self-administered questionnaire, to collect data on their socio-demographic, behavioral and life style characteristics, and diagnostic questions following Rome III criteria to assess IBS occurrence. The difference in IBS prevalence by socio-demographic characteristics, smoking, alcohol consumption, and physical activity was assessed by using the Chi-square test. Logistic regression adjusted odds ratios were used to investigate the association between risk factors and IBS. Results Data was collected from 553 individuals and consisted of 52.8% females (mean age 35.9 years, SD = 11.9) and 47.2% males (mean age = 36.1 years, SD = 10.3). The prevalence of IBS in the study population according to Rome III criteria was 20.1%. The bivariate analysis indicated that being younger than 30 years old, a female, an ever water pipe smoker, an ever alcohol consumer are significantly associated with a higher prevalence of IBS. Educational level, cigarettes smoking and physical exercise were not significantly associated with IBS occurrence. The logistic regression adjusted odds ratio showed that females were 1.67 times more likely to have IBS than males (P˂ 0.05). The participants aged less than 30 years old were at a higher risk of having IBS (P˂ 0.01). Those who ever smoked waterpipe were 1.63 times more likely to have IBS than those who never smoked waterpipe (P˂ 0.05). Those who were ever alcohol drinkers were twice as likely to have IBS than never-drinkers (P˂ 0.01). Conclusion New data on the high prevalence of IBS in an adult population in Lebanon has been reported. This is also the first study to investigate and show an association of waterpipe smoking and IBS. Further longitudinal studies are warranted to determine whether this association is causal

Additional file 1: of Irritable bowel syndrome: prevalence, risk factors in an adult Lebanese population

Bloating Questionnaire. (DOCX 98 kb

An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis