title: Freeze substitution and freeze drying for stable, long-term preservation of cytologic specimens for immunostaining.
abstract: OBJECTIVE
  To develop a new method of fixing and preserving cytologic specimens for immunostaining after long-term storage at room temperature.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  Freeze substitution and freeze drying are an alternative method of preserving cytologic specimens.
authors.affiliation: Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Takahashi
authors.forename: T
authors.initials: T
authors.lastname: Okuyama
authors.forename: S
authors.initials: S
authors.lastname: Matsuo
authors.forename: S
authors.initials: S
authors.lastname: Maeda
mesh_terms: D018288:Carcinoma, Small Cell / Q000175:diagnosis; D003584:Cytological Techniques; D003890:Desiccation; D005612:Freeze Drying / Q000379:methods*; D017110:Freeze Substitution / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D009584:Nitrogen; D010100:Oxygen; D013048:Specimen Handling / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation / Q000379:methods; D014407:Tumor Cells, Cultured / Q000166:cytology
chemical_list: D009584:Nitrogen; D010100:Oxygen