Laser microdissection combined with immunohistochemistry on serial thin tissue sections: a method allowing efficient mRNA analysis.
Laser microdissection (LMD) with subsequent reverse transcription-PCR analysis is a powerful histochemical technique subserving the molecular characterization of specific cell types. We developed an efficient method for selective sampling of specific cell populations using immunohistochemistry coupled with LMD. The cerebral cortex of adult rats was cut into serial thin sections. Some sections were immunostained for parvalbumin. The adjacent sections were mounted on Cell Support Film for LMD and stained with neutral red. By comparison of the two adjacent sections, neuronal profiles representing parts of parvalbumin-immunopositive somata were identified in the neutral red-stained sections. These neuronal profiles were safely captured with LMD and analyzed on reverse transcription-PCR using extracted RNA. The method presented here can be applied to cell-type-specific characterizations using fixed cells under RNase-free conditions.
Kase
Masahiko
M
Department of Anatomy and Brain Science, Kansai Medical University, Moriguchi, Osaka, 570-8506, Japan.
Houtani
Takeshi
T
Sakuma
Satoru
S
Tsutsumi
Toshiyuki
T
Sugimoto
Tetsuo
T
D000818:Animals; D002540:Cerebral Cortex / Q000737:chemistry* / Q000166:cytology; D005629:Frozen Sections; D016591:Histocytological Preparation Techniques; D007150:Immunohistochemistry / Q000379:methods*; D007834:Lasers; D008297:Male; D042282:Microdissection / Q000379:methods*; D008846:Micromanipulation; D009474:Neurons / Q000737:chemistry*; D010320:Parvalbumins / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley
D010320:Parvalbumins; D012333:RNA, Messenger