RESUMEN
In situ hybridization for the detection of RNAm in ultrathin tissue sections embedded in lowicryl resin is reported. This method proved suitable for both tissues and cell cultures, using either complementary DNA probes or synthetic oligonucleotides, labeled with a radioisotope or biotin. This method seems to strike a satisfactory balance between preservation of cell ultrastructure and sensitivity.
Asunto(s)
Resinas Acrílicas , Hibridación in Situ/métodos , ARN Mensajero/análisis , Animales , Neoplasias de la Mama/genética , Receptores ErbB/genética , Femenino , Hormona del Crecimiento/genética , Humanos , Técnicas In Vitro , Masculino , Microscopía Electrónica , ARN Mensajero/ultraestructura , Ratas , Ratas WistarRESUMEN
In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultra-thin frozen sections, or on ultra-thin sections of tissues embedded in hydrophilic resin such as Lowicryl. With the purpose of comparing the sensitivity, resolution, and ultrastructural preservation of these three methods, we examined the expression of the growth hormone (GH) gene in anterior pituitary cells by in situ hybridization at the ultrastructural level, using a synthetic oligonucleotide complementary to the codons of the mRNA from Gln 45 to Ser 54 labeled at the 3' end of biotin-21dUTP. All these methods gave similar results: mRNA was located on the lamellar endoplasmic reticulum of somatotrophs. The pre-embedding method gave the best ultrastructural preservation, with low resolution with the enzymatic detection system and an intermediate sensitivity. A probe concentration of 10 pmol/ml was sufficient to obtain a signal. With this method gold particles could not be used without pre-treatment. The frozen section method gave the best sensitivity (a signal was observed with 4 pmol/ml of probe) but the lowest ultrastructural preservation. On ultra-thin Lowicryl sections, resolution was as high as with the frozen-section method, ultrastructural conservation was intermediate, and sensitivity was low. These results indicate that the last method seems to be a good compromise between sensitivity and ultrastructural preservation.