Recovery of nuclei from glycol-methacrylate-embedded tissue
ET Wright, JW Jacobberger, TP Pretlow and TG Pretlow
Department of Pathology, Case Western Reserve University, Cleveland, OH
44106.
The analysis of antigens, enzyme histochemical markers, and DNA has become
an important part of the classification of some leukemias, lymphomas, and
other neoplastic diseases. Many of the relevant antigens and most of the
relevant enzyme histochemical activities are destroyed and others are less
than optimally preserved in tissues embedded in hot paraffin. Most
enzymatic activities and antigens are well preserved in tissues embedded at
4 degrees C in glycol methacrylate (GMA). The measurement of DNA content in
neoplastic cells with the most commonly employed techniques depended on the
availability of fresh suspensions of cells until the development by Hedley
of methods that permit the recovery of nuclei from paraffin blocks for this
purpose. In order to facilitate the analysis of antigens, enzymatic
markers, and DNA from the same sample of tissue, we have developed a means
of recovery of nuclei from GMA-embedded tissues. Twenty-microns-thick
sections of GMA- embedded tonsil were either pretreated with an organic
solvent (absolute ethanol or 2-ethoxyethanol) followed by rehydration in
phosphate buffered saline (PBS) or directly rehydrated in PBS. The
suspensions were formed mechanically by gentle sonication. The type of
fixative and length of PBS rehydration were varied. Tissue fixed in 100%
acetone, embedded in GMA, and rehydrated directly in PBS for six days gave
the highest average yield of nuclei, 3.7 x 10(7) nuclei per gram tissue. In
order to assess DNA binding of fluorescent dyes, 2- microns-thick GMA
sections were stained with chromomycin, Hoechst 33342 (Sigma Chemical, St
Louis, MO), and propidium iodide. Hoechst 33342 bound specifically to the
nuclei with low background staining.
Volume 76,
Issue 8,
pp. 1622-1625,
10/15/1990
Copyright © 1990 by The American Society of Hematology
