Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples.
Proteomic analysis of the brain is complicated by the need to obtain cells from specific anatomical regions, or nuclei. Laser capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section, and thus could be useful for isolating specific brain nuclei for analysis. However, we and others have previously demonstrated that histological staining protocols used to guide LCM have detrimental effects on protein separation by two-dimensional electrophoresis (2-DE). Here we describe a new LCM method called navigated LCM. This microdissection method uses fixed but unstained tissue as starting material and thus enables us to avoid artifacts induced by tissue staining. By comparing 2-DE results obtained from fixed, unstained LCM brain tissue samples to those obtained from manually dissected samples, we demonstrated that this microdissection process gave similar protein recovery rates and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of selected spots from gels derived from control and fixed, LCM samples revealed that the fixation-LCM process had no effect on protein identification. Navigated LCM of tissue sections is therefore a practical and powerful method for performing proteomic studies in specifically defined brain regions.
Department of Anesthesiology, UT-MD Anderson Cancer Center, Houston, TX 77030, USA.
Lionel
L
Moulédous
Sybille
S
Hunt
Rebecca
R
Harcourt
Jenny
J
Harry
Keith L
KL
Williams
Howard B
HB
Gutstein
D000818:Animals; D001921:Brain / Q000033:anatomy & histology* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D008297:Male; D042282:Microdissection / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013194:Staining and Labeling
D009419:Nerve Tissue Proteins