Optimizing staining protocols for laser microdissection of specific cell types from the testis including carcinoma in situ.
Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdissection technology allows for enrichment of specific cell types. However, when the cells are not morphologically distinguishable, it is necessary to use a specific staining method for the target cells. In this study we have tested different fixatives, storage conditions for frozen sections and staining protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet-containing cells, which is useful for isolation of the androgen-producing Leydig cells. Both protocols retain a morphology that is compatible with laser microdissection and yield RNA of a quality suitable for PCR and microarray analysis.
Sonne
Si Brask
SB
Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark. si@bsonne.dk
Dalgaard
Marlene D
MD
Nielsen
John Erik
JE
Hoei-Hansen
Christina E
CE
Rajpert-De Meyts
Ewa
E
Gjerdrum
Lise Mette
LM
Leffers
Henrik
H
D002278:Carcinoma in Situ / Q000473:pathology*; D006801:Humans; D007834:Lasers; D008297:Male; D046228:Microarray Analysis; D042282:Microdissection / Q000295:instrumentation* / Q000379:methods; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D012334:RNA, Neoplasm / Q000302:isolation & purification / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D013736:Testicular Neoplasms / Q000473:pathology*; D013737:Testis / Q000166:cytology* / Q000378:metabolism
D012334:RNA, Neoplasm; D012313:RNA