Laser capture microdissection--a demonstration of the isolation of individual dopamine neurons and the entire ventral tegmental area.
Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.
Department of Basic Sciences, Mississippi State University College of Veterinary Medicine.
Evangel
E
Kummari
Department of Basic Sciences, Mississippi State University College of Veterinary Medicine.
Shirley X
SX
Guo-Ross
Department of Basic Sciences, Mississippi State University College of Veterinary Medicine; eells@cvm.msstate.edu.
Jeffrey B
JB
Eells
D000818:Animals; D059290:Dopaminergic Neurons / Q000166:cytology*; D007150:Immunohistochemistry; D061346:Laser Capture Microdissection / Q000379:methods*; D051379:Mice; D017557:Ventral Tegmental Area / Q000166:cytology*