Influence of four different freezing methods on quality of frozen sections of mouse brain tissues

MENG Jian; FAN Hongguang

doi:10.3969/j.issn.1002-266X.2025.05.011

Abstract：Objective To compare the effects of four different freezing methods on the quality of frozen sections of mouse brain tissues.Methods Twenty samples of brain tissues from 4-month-old wild-type male mice(C57 mice)were selected and frozen sections were prepared using four methods:-80℃rapid freezing,isopentane-liquid nitrogen rapid freezing method,isopentane-liquid nitrogen rapid freezing,liquid nitrogen rapid freezing,and formaldehyde fixa-tion-gradient sucrose(with concentrations of 20%and 30%)dehydration freezing.Five samples were prepared for each method.The sections were stained with HE and the morphology and structure of the cells were observed under the micro-scope.Results The quality of frozen sections prepared by-80℃rapid freezing,liquid nitrogen rapid freezing,and formaldehyde fixation-gradient sucrose(with concentrations of 20%and 30%)dehydration freezing methods was generally poor.Large area of ice crystals was visible under the microscope,causing cell cytoplasmic atrophy and presenting a cord-like destruction,and the intercellular space was enlarged.Compared with the other methods,the quality of frozen sections prepared by isopentane-liquid nitrogen rapid freezing was better.Under the microscope,the brain tissue structure was com-plete,the cell staining was clear,the cell boundaries were distinct,the nucleus and cytoplasm were clearly separated,the contrast was good,and there were no knife marks,wrinkles,overlaps,or ice crystals.The sections were flat and of uni-form thickness.Conclusion Compared with-80℃rapid freezing,liquid nitrogen rapid freezing,and formaldehyde fix-ation-gradient sucrose(with concentrations of 20%and 30%)dehydration freezing,the quality of frozen sections of mouse brain tissues prepared by isopentane-liquid nitrogen rapid freezing was the best.