The Retention of Water-soluble Compounds during Freeze-Substitution and Microautoradiography

Abstract

Freeze-substitution and Epon embedment were quantitatively evaluated for their effectiveness in retaining water-soluble metabolites in plant tissues. Roughly 99% of the 80% (v/v) ethanol-extractable radioactivity in photosynthetically labeled soybean leaf discs and in petiole fragments containing translocated ¹⁴C was retained during freeze-substitution in acetone or propylene oxide and embedment in Epon. Substantially more activity was lost from ¹⁴C-sucrose-infiltrated pith blocks, but most or all of this loss came from the block surface. The procedure was effective for a sucrose concentration as low as 0.004%. Sections floated on water retained most of their ¹⁴C-sucrose, and high resolution autoradiographs could easily be prepared without resorting to dry procedures. Embedded ¹⁴C-sucrose was apparently chemically unreactive, since there was no loss of radioactivity when sections were stained with the periodic acid-Schiff reagent, nor did the embedded sucrose show staining.