ABSTRACT
We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.
