Cryoprotectant Tissue Storage Solutions: Stability at Lower Temperatures, Longer Storage Times, More Versatile Usage

Abstract

Abstract Infusion with buffer/sucrose solutions (up to 30% sucrose) has long been used to ‘cryoprotect’ tissue in an attempt to prevent ice crystal artifact in frozen sections. This is helpful for example in sectioning large, fixed tissue blocks that must be frozen relatively slowly in dry ice to allow sectioning on a sliding microtome. In 1977, De Olmos added ethylene glycol to the mixture (30 g cane sugar/50 ml 0.1 M PO4 buffer at pH 7.2 in 20 ml ethylene glycoi) for -10°C storage of free floating sections from lightly fixed primate brain. Jones and Kane in 78 used this solution for storage of sections at -20°C (standard household freezer temperature) for up to one month before horseradish peroxidase histochemical reaction, in their methods, they cautioned that "sucrose attracts insects" (ants, personal communication). We and others have found the above cryoprotectant solution generally useful for storage of free floating sections from fixed brain. We observed that adjacent sections stored at 4°C in buffer for 2 weeks (common practice for Nissl stained sections) lost their reactivity to antibody labelling.