Lack of compatibility of histological staining methods with proteomic analysis of laser-capture microdissected brain samples.
The anatomical complexity of the brain presents a challenge for the analysis of changes in gene and protein expression. Laser-capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section. Protein expression in tissues obtained by LCM has been studied by Western blot and two-dimensional (2D) gel electrophoresis. However, it is not known whether histological staining techniques interfere with protein recovery and resolution on 2D gels. The goal of this study was to determine the effects of staining procedures on protein extraction and separation. LCM samples of rat brain striatum obtained after histological staining were compared with unstained, LCM-captured tissue and fresh-frozen, unstained, manually dissected samples. Specimens subsequently underwent protein extraction and 2D gel electrophoresis under identical conditions. Our results indicated that histological staining of the tissue greatly reduced protein recovery from LCM-captured samples. However, fixation and LCM without histological staining did not significantly affect protein recovery from brain tissue. These results indicate that LCM of fixed, unstained brain tissue could be used to dissect discrete brain regions for proteomic analysis. Histological staining of neural tissue should be avoided, because it interferes with protein recovery. LCM appears to be a promising tool for the study of localized protein changes underlying brain plasticity.
Mouledous
Lionel
L
Department of Anesthesiology, M. D. Anderson Cancer Center, Houston, TX 77030, USA.
Hunt
Sybille
S
Harcourt
Rebecca
R
Harry
Jenny L
JL
Williams
Keith L
KL
Gutstein
Howard B
HB