A method for immunofluorescent localization of oestrogen receptors in bone sections from an egg-laying poultry strain

Abstract

Although oestrogen has profound skeletal effects in hens, the identity of its target cells in bone is still unclear. We wished to address this by indirect immunofluorescent detection of oestrogen receptors, using monoclonal antibodies, similar to our method for mammalian bone. Avian bone, however, is prone to autofluorescence at the excitation wavelength for fluorescein isothiocyanate, and non-specific binding of mammalian antibodies. We therefore improved receptor detection by comparing three commercially available monoclonal antibodies to the human oestrogen receptor. We found that the best identification of oestrogen target cells was produced by ID5 antibody diluted 1/20, with initial binding disclosed by Cy3a(TM)-conjugated immunoglobin, which has similar fluorescence to rhodamine. Clear localisation of these cells was reliably obtained in sections of both receptor positive human breast tissue and hen oviduct. Preliminary observations showed that immunofluorescence in avian oviduct and undecalcified bone cryosections was stable after 6 weeks storage and of sufficient clarity for semiquantification. Thus, in hens aged 18 weeks (first ovarian follicle), osteoblasts and 38% of osteocytes were clearly immunofluorescent. After 8 to 10 weeks egg lay, receptor-positive osteocytes decreased in structural bone to 19%; cells adjacent to medullary bone and in marrow cavities were strongly immunofluorescent.