We performed glutathione (GSH) staining at a low temperature to prevent GSH release from the section and, hence, improve morphology. Fresh frozen sections of liver, lung, kidney, heart, and stomach were incubated for GSH activity in an ice bath (2-5°C) for 5-10 min. Low temperature incubation prevented GSH diffusion out of cells and minimized migration of granules into vessels and outside of tissue. Incubation at low temparature generally reduced the intensity of the stain compared to the standard method. We conclude that low temperature incubation improves GSH localization in cells, probably by regulating the rate, formation, and the size of GSH-mercury orange complexes.
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