Modern acrylics for post-embedding immunostaining techniques

Abstract

We describe two methods for rapid processing of biological tissues into LR White acrylic plastic. Both methods make use of LR White's compatibility with small amounts of water, enabling non-osmicated tissue to be only partially dehydrated before infiltration with the plastic, a procedure that improves the sensitivity of post-embedding immunocytochemistry. In addition, both methods are designed to reduce the time for which tissue is exposed to the damaging influence of the plastic monomer, which can cause extraction and sudden shrinkage. The tissue example used in the first method is immersion-fixed, surgically removed human pituitary which, by virtue of its thorough fixation, can be processed quickly at 50 degrees C using catalytic polymerization at room temperature. The concentration of the catalyst is critically set to prevent the temperature rising above 60 degrees C in the tissue blocks. Penetration of immunoperoxidase reagents into 330-nm LR White sections is demonstrated and possible modes of action are discussed. When "lightly" fixed tissue is processed as above, serious polymerization artifacts can result from autocatalysis. A second method, based on the first but employing slower polymerization at 0 degrees C, has therefore been developed. The high level of fine structure that can be retained using this method is illustrated by the demonstration of the trans-tubular Golgi in perfusion-fixed kidney of rat. Biotinylated lectin is localized to cells of the kidney proximal tubule with streptavidin-colloidal gold, to illustrate tissue reactivity. In a second example, the structure of the bacterial cell envelope is shown to be similar in appearance after partial dehydration and LR White embedding to that seen after progressive lowering of temperature, dehydration, and Lowicryl embedding.