Improved method for release of cell nuclei from paraffin-embedded cell material of squamous cell carcinomas.
An improved method of releasing cell nuclei from paraffin-embedded highly keratinized squamous cell carcinomas by pretreatment with 85% formic acid-0.3% H2O2 followed by enzymatic treatment with subtilisin Carlsberg is described. After DAPI staining the resulting suspensions of cell nuclei were analyzed by DNA flow cytometry, in addition to microscopy. The total yield of released cell nuclei was improved and the proportion of tumor cell nuclei in the suspensions increased from 12% to 53% using this method compared to the conventional preparation technique. Cytoplasmic residue disappeared nearly completely. Thus, the finding of false aneuploid cell populations representing diploid cells with autofluorescent cytoplasm could be avoided. In addition, even small true aneuploid cell populations could be detected due to the increased proportion of released tumor cell nuclei and the lower proportion of background in the > 2C region.
Wang
N
N
Department of Medical Radiobiology, Karolinska Institute, Stockholm, Sweden.
Pan
Y
Y
Heiden
T
T
Tribukait
B
B
D002294:Carcinoma, Squamous Cell / Q000473:pathology* / Q000648:ultrastructure; D002467:Cell Nucleus / Q000648:ultrastructure*; D004220:Disulfides / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005561:Formates / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D010232:Paraffin; D013381:Subtilisins; D001749:Urinary Bladder Neoplasms / Q000473:pathology* / Q000648:ultrastructure
D004220:Disulfides; D005561:Formates; D010232:Paraffin; D006861:Hydrogen Peroxide; D013381:Subtilisins