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.
Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark. si@bsonne.dk
Si Brask
SB
Sonne
Marlene D
MD
Dalgaard
John Erik
JE
Nielsen
Christina E
CE
Hoei-Hansen
Ewa
E
Rajpert-De Meyts
Lise Mette
LM
Gjerdrum
Henrik
H
Leffers
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