On the same model, we investi gated a possible relationship between COX 2 pathway and osteogenic differentiation by analyzing mRNA http://www.selleckchem.com/products/Romidepsin-FK228.html b ALP expression in presence or absence of COX 2 inhibitor. Materials and methods Animals Forty six month old female Sprague Dawley rats of approximately 200 to 300 g body weight were obtained from Charles River Italia. All rats were housed under similar conditions. They were fed a stan dard rodent diet containing 0. 97% calcium, 0. 85% phos phorus, 1,045 IU Kg vitamin D3, 22. 5% protein, 5. 5% fat, 52% carbohydrate, and were given access to tap water ad libitum. The animal procedures were approved by the local government authorities and were conducted in accord with accepted standards of humane animal care, as out lined in the Ethical Guidelines.

Drugs Risedronate in powder form was provided by Proc ter and Gamble Pharmaceuticals Inhibitors,Modulators,Libraries Inc. Ris was dissolved in deionized water and was administered at 5 ug Kg subcutaneously three times a week. Methylprednisolone was diluted Inhibitors,Modulators,Libraries in a sesame oil vehicle at a concentration of 7 mg kg. The right tibiae were removed, dissected free of soft tis sue, and fixed in 70% reagent alcohol. The samples were embedded undecalcified in methyl methacrylate resin. Measurements were performed by means of an image analysis system consisting of an epifluorescent micro scope connected to an analogic 3 CCD camera and a computer equipped with a specific software for histomorphometric analyses. The area analyzed was restricted to the trabecular bone of the secondary spongiosa area between 2 and 4 mm dis tal to the growth plate metaphyseal junction.

Histomorphometric parameters are reported in accor dance with the ASBMR Committee nomenclature. Thickness Inhibitors,Modulators,Libraries results Inhibitors,Modulators,Libraries were adjusted for the obliquity of sections by multiplying by �� 4. Immunohistochemical determination of COX 2 expression Explants of left femora were fixed in 4% buffer formalin for 24 hours, decalcified with 0. 5 mol L ethylendiaminete traacetic acid, pH 8, for 7 to 10 days, paraffin embedded, and cross sectioned at three different levels. Goat Serum was purchased from Sigma Aldrich Co. Primary Antibody, rabbit anti mouse cox 2, from NeoMarkers Inc. Sec ondary Antibody, goat anti rabbit IgG B, from SantaCruz Biotechnology Inc. Santa Cruz, CA, USA, ABC solution, Vectastain ABC kit, from Vector Laboratories Inc.

DAB solution, Liquid DAB Substrate Chromogen System, from DakoCytomation. Immunohystochemistry was performed on paraffin embebbed specimen sections, following the technique of Fortier et al. with the same modifica tion. Inhibitors,Modulators,Libraries Briefly, slides were stored in the stove at 60 C till paraffin loosed, and then were washed in xylene, followed by rehydration through graded ethanol washes. Permeabi lization was performed selleck chemical by heating at 70 in a humidified chamber, the sections were previously covered with 10 mM citrate buffer, pH 6. 0, followed by cooling at room temperature.