Laser microdissection and pressure catapulting: combining LMPC with IHC to investigate NMDA receptor subunit composition.
Laser microdissection and pressure catapulting (LMPC) has allowed investigators the opportunity to look at enriched cell populations from heterogeneous tissues. Selection of cells is routinely based upon morphological or histological criteria. The inclusion of an immunohistochemistry step prior to LMPC potentially allows investigators to classify cell populations both morphologically and functionally. Practical limitations with regard to RNA integrity following IHC steps have meant that the value that this combination brings has been reduced. Here, through the use of RNA preserving buffers, we have been able to successfully label, dissect and extract RNA from NK1 expressing cells. RT-PCR was carried out to confirm the expression of NK1, NR2A and NR2B subunit expression. Our data confirmed the expression of NK1 in cells labelled with the antibody, and the absence of expression in cells absent of staining. As well as this, relative expression of both NR2A and NR2B was determined. Furthermore, the RNA was of high enough quality to allow these methods to be used for studies involving RNA amplification steps, such as microarray analysis.
Brinkmann
Hannah
H
Drug Safety Research and Development, Pfizer Inc., Ramsgate Road, Sandwich CT13 9NJ, UK. hannah.brinkmann@pfizer.com
Curry
John Aloysius
JA
D000199:Actins / Q000096:biosynthesis; D000818:Animals; D003584:Cytological Techniques; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D042282:Microdissection / Q000379:methods*; D011312:Pressure; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D018076:DNA, Complementary; C473649:NR1A NMDA receptor, rat; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; D016194:Receptors, N-Methyl-D-Aspartate; D012313:RNA