Estabelecimento do protocolo Agnor para membranas embrionárias e fetais de equinos

Abstract

The nucleolus organizing regions (RONs) or Nucleolar Organizer Regions (NORs) are slightly stained chromatin regions around which, at the end of the telophase, the nucleolus is formed again after mitosis. A peculiar group of acidic proteins that have a high affinity for silver, are located in the same places as NORs, which gives the same characteristics of being clearly and quickly visualized by stains using silver nitrate. For this study, equine embryo / fetus membranes obtained from 30 placentas were used. Such material was collected from adult, mixed breed mares and at different gestation periods of up to 100 days. Fragments of the embryo / fetus membranes were fixed in 10% formaldehyde for routine histological procedures (TOLOSA et al, 2003). Serial cuts with a thickness of 5μm were obtained in a microtome (Leica 2165®,) which were dewaxed and immersed in a final solution 1: 2, consisting of colloidal solution (gelatin 2% and formic acid 1%) and silver nitrate solution (AgNO3) (1: 1). They were taken to the oven at 37 ° C, in a humid chamber, for approximately 20 minutes. After washing the slides with water, a 1% sodium thiosulfate solution was applied for one minute and followed by washing and drying at room temperature, being stored in a darkroom. After the silver impregnation staining of the NORs (Nucleolar Organizer Regions) some slides were subjected to Eosin baths so that the cytoplasm was stained and thus obtaining easier cell individualization. The results obtained were appropriate, showing the appropriate protocol, and the eosin background provided a better visualization of cell delimitations, thus facilitating the possible counting of NORs per cell.