Freeze substitution and freeze drying for stable, long-term preservation of cytologic specimens for immunostaining

To develop a new method of fixing and preserving cytologic specimens for immunostaining after long-term storage at room temperature.

The method consists of three steps: fixation, freeze substitution or freeze drying, and storage. To test the method, we used the human small cell lung carcinoma cell line Lu-135, which expresses a high level of mutant p53 protein and exhibits strong nuclear staining when reacted with an anti-p53 antibody. A smear of Lu-135 cells was fixed in a mixture of methanol and ether (50:50=vol/vol) or sprayed with a fixative containing isopropyl alcohol, methanol and polyethylene glycol. The fixed cells were freeze substituted by immersing them in a dry ice/methanol/ether bath and then were freeze dried under a vacuum. The smear was then placed in a 50-mL conical tube containing silica gel. The tube was sealed and stored at room temperature.

Freeze substituted cells that were fixed with methanol/ether and stored for more than six months retained strong p53 positivity, as strong as that of the control cells, which had been fixed and stored in methanol.

Freeze substitution and freeze drying are an alternative method of preserving cytologic specimens.