Preparation of human heart for laser microdissection and proteomics.
Proteomics generates information on expressed proteins, and laser microdissection (LMD) is a method that allows enrichment of specific cell types from complex heterogeneous tissue. Together they provide a powerful tool for functional genomic research. Here, we have investigated (i) the effects of fixation and staining on cardiac proteins separated by two-dimensional gel electrophoresis (2-DE) and (ii) feasibility of using LMD to separately prepare myocytes and blood vessels for 2-DE gel analysis. This is the first such study of human heart. The effect of fixation (ethanol or acetone), staining with haematoxylin and eosin in the presence and absence of xylene, and antibody staining was investigated. Proteins were separated by 2-DE and spots detected by silver staining. Quantitative spot analysis showed that contractile proteins were preserved under all conditions, and no significant differences were found when the groups studied were compared with the control group. However, there were differences in the visual quality of the gel patterns. LMD provided enough protein from blood vessels and myocytes to run one large-format (18 x 24 cm) 2-D gel for each subset of cells. Collection of this material took 70 h (approximately 2800 blood vessels and 17,000 myocytes) and resulted in tissue-specific gel patterns for these two structures. In conclusion, the use of haematoxylin and eosin staining without xylene provided the best morphology and did not significantly affect protein spot number.
National Heart and Lung Institute, Imperial College, London.
Ayesha I
AI
De Souza
Emma
E
McGregor
Michael J
MJ
Dunn
Marlene L
ML
Rose
D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006321:Heart; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007834:Lasers; D009206:Myocardium / Q000166:cytology / Q000378:metabolism / Q000473:pathology*; D011506:Proteins / Q000032:analysis; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D016622:Silver Staining; D014992:Xylenes / Q000494:pharmacology
D011506:Proteins; D020543:Proteome; D014992:Xylenes