5 mg protein. Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.2% bovine serum albumin (BSA) was then added to make up the total assay volume of 250 µl. The required protein concentration and incubation period were determined by the standard curve using various

Selleck GDC0199 concentrations. After incubation in a water bath at 37ºC for two hours, the reaction was terminated by the addition of ethyl acetate Inhibitors,research,lifescience,medical and the steroids were then extracted. The organic layer was separated by centrifugation at 3000 rpm and 4ºC for 10 min. The top layer was then transferred into new test tubes and evaporated to dryness at 55ºC in a vacuum concentrator. Steroid residues were dissolved in an ethanol containing nonradioactive carrier of 11-dehydrocorticosterone and corticosterone. Inhibitors,research,lifescience,medical They were then separated by thin layer chromatography, TLC (Whatman, UK) in 92:8 ratio of chloroform and 95% ethanol. The fractions corresponding to the steroids were located by UV lamp absorption at 240 nm, scraped, transferred into scintillation vials and counted Inhibitors,research,lifescience,medical in scintillation fluid (Cocktail T) in a Kontron Betamatic fluid scintillation counter (Merck, Germany). Enzyme activity was calculated as the percentage conversion of the active [3H] corticosterone to inactive [3H] 11-dehydrocorticosterone from the radioactivity of each fraction. Enzyme

activity was measured by the method used by previous studies with some modification.19,20 The statistical software used was the Statistical Package for Social Sciences (SPSS) version Inhibitors,research,lifescience,medical 12. The data was tested for normality using

the Kolmogrov-Smirnov test. Since the data were found to be normally distributed, they were analyzed using one-way Analysis of Variance (ANOVA) followed by Tukey pos-hoc test for pairwise comparisons A P value of <0.05 were taken as significant. Data are presented as mean±standard error of the means (SEM). Measurement of 11β-HSD1 Expression The formalin-fixed paraffin-embedded Inhibitors,research,lifescience,medical bone sections were deparaffinized and rehydrated. For antigen retrieval, the sections were incubated in 0.01M citrate buffer at 90ºC for 5 minutes. The sections then were incubated in 0.3% hydrogen peroxide for 10 minutes to block the endogenous peroxide activity and subsequently incubated in 1:50 normal goat serum (Vector Laboratories; Burlingame, CA) for 20 minutes to block nonspecific antibody binding. SB-3CT Sections were incubated for 60 minutes with primary rabbit 11β-Hydroxysteroid Dehydrogenase (Type1) Polyclonal Antitbody, and detected by goat anti-rabbit peroxidase (Vector Laboratories, Burlinghame California) using DAB as a chromogen according to the manufacture’s instructions and counterstained with haematoxylin. Controls were done by using positive tissues (liver and adipose tissue) and omissions of the primary antibody. Photomicrographs taken were scored by two blind observers at 25 and 50 times magnification.