Laser capture microdissection and molecular analysis of Plasmodium yoelii liver-stage parasites.
Laser capture microdissection and molecular analysis of Plasmodium yoelii liver-stage parasites.
复制标题
约氏疟原虫肝期寄生虫的激光捕获显微切割和分子分析。
DOI：
10.1016/s0166-6851(01)00411-x
复制
发表时间：
2002
影响因子：
1.5
通讯作者：
Hoffman,StephenL
中科院分区：
文献类型：
--
作者：
SacciJr,JohnB;Aguiar,JoaoC;Lau,AudreyO;Hoffman,StephenL
The application of the laser capture microdissection (LCM) in the isolation ofP. yoeliiliver stage schizonts and the molecular characterization of the isolated parasites by reverse transcriptase-polymerase chain reaction and Western blotting were described. BALB/c mice received an intravenous injection in the tail of 5×106P. yoelii, gradient isolated, sporozoites were euthanized by CO2overdose 44 h postinfection and their livers were removed for cryosectioning or RNA extraction. The large inoculum of sporozoites injected into the mice yielded a heavily infected liver containing numerous schizonts at 44 h postinfection. LCM of liver stage schizonts, from cryosections, was accomplished using a Pixcell laser capture microscope. Cryosections utilized for LCM allowed the clear separation of liver schizonts from the surrounding hepatocytes. In virtually all cases, the entire schizont was recovered, with little or none of the surrounding hepatocytes. Approximately 500 captured liver-stage schizonts were solubilized by sequential incubations of the LCM caps with 20 µl of SDS-PAGE sample buffer. The samples were assessed by Western blot analysis, utilizing the monoclonal antibody navy yoelii liver stage 3 (NYLS3). A single band with a molecular weight of ∼17 kDa was identified in the protein extracts from LCM schizonts.
