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Optimization of trypsin treatment condition utilizing immunohistochemistry for chromogenic in situ hybridization
Author(s) -
Nakamura Nobuyuki,
Fujii Takeshi,
Soejima Yurie,
Sawabe Motoji
Publication year - 2019
Publication title -
pathology international
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.73
H-Index - 74
eISSN - 1440-1827
pISSN - 1320-5463
DOI - 10.1111/pin.12855
Subject(s) - cish , trypsin , protease , chromogenic in situ hybridization , anaplastic lymphoma kinase , microbiology and biotechnology , in situ hybridization , histone , chemistry , biology , biochemistry , cancer research , enzyme , pathology , gene , medicine , gene expression , malignant pleural effusion , lung cancer
Chromogenic in situ hybridization (CISH) is a molecular technique used to visualize specific genes. Both heat treatment and protease treatment play important roles for the success of CISH on formalin‐fixed paraffin‐embedded (FFPE) tissue sections. In contrast to heat treatment, the optimal condition of protease treatment may vary depending on each sample. Because trypsin has a substrate specificity to cleave lysine and arginine, we hypothesized that trypsin could effectively degrade histones rich in lysine and arginine and that the removal of histones from DNA following heat treatment could improve CISH results. We selected 21 patients with lung adenocarcinoma previously known to be positive or negative for anaplastic lymphoma kinase ( ALK ) gene rearrangement and used FFPE tissue sections collected from these patients. Then, we assessed histone degradation among the following protease treatments; trypsin, pepsin, and proteinase K, and compared the ALK CISH results with results obtained using commercially available kits and these protease treatments. The results showed that trypsin effectively degraded histones. Additionally, compared with the other treatments, ALK CISH with trypsin treatment showed the most evaluable cells and the smallest standard deviation. Our study suggests that the degradation of histones by trypsin subsequent to heat treatment might improve CISH results.