As the brain of Patient H.M. was cut, histological slices were being collected in serial order in 36-well compartment boxes containing 0.1 M phosphate buffer solution. The next step is to store all tissue slices long-term in the freezer in a solution that will preserve the morphology and molecular milieu of the tissue.
In the next 10 days, we will move tissue slices into cryo-storage vials that have been individually numbered, filled with: Phosphate Buffer, Ethylene glycol, and Polyvinylpyrrolidone (PVP). As Dr. Switzer of Neuroscience Associates correctly points out, this solution is not meant as a cryo-protective but it preserves the antigenicity of proteins (for subsequent immune-cytochemistry). Nevertheless, Ethylene glycol effectively prevents ice crystal formation at temperatures below 0°C. Reference: Use of cryo-protectant to maintain long-term peptide immune-reactivity and tissue morphology Robert E. Watson, Jr., Stanley J. Wiegand, Richard W. Clough, Gloria E. Hoffman. Peptides 1986;7:155-159.
This protocol should guarantee that the quality of tissue sections is preserved during the time it will take to stain and account for the entire series.