Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification.
An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.
Bi
Wenya Linda
WL
Massachusetts General Hospital, Charlestown, MA, USA.
Keller-McGandy
Christine
C
Standaert
David G
DG
Augood
Sarah J
SJ
D000818:Animals; D002232:Carbocyanines / Q000032:analysis; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D003199:Computer Systems; D003342:Corpus Striatum / Q000166:cytology / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis*; D005629:Frozen Sections; D006834:Hydrazines / Q000032:analysis; D007395:Interneurons / Q000201:enzymology*; D007527:Isoenzymes / Q000032:analysis; D007834:Lasers; D009419:Nerve Tissue Proteins / Q000032:analysis*; D019001:Nitric Oxide Synthase / Q000032:analysis*; D052248:Nitric Oxide Synthase Type I; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000235:genetics*; D012235:Rhodamines / Q000032:analysis; D013048:Specimen Handling; D013997:Time Factors
C417561:Alexa 488 hydrazide; D002232:Carbocyanines; D005456:Fluorescent Dyes; D006834:Hydrazines; D007527:Isoenzymes; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; C464436:NR2D NMDA receptor; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D012235:Rhodamines; C089187:cyanine dye 3; C011203:cyanine dye 2; C005358:tetramethylrhodamine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat