Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

TY - JOUR

T1 - Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

AU - Sonne, Si Brask

AU - Dalgaard, Marlene D

AU - Nielsen, John Erik

AU - Hoei-Hansen, Christina E

AU - Rajpert-De Meyts, Ewa

AU - Gjerdrum, Lise Mette

AU - Leffers, Henrik

N1 - Keywords: Carcinoma in Situ; Humans; Lasers; Male; Microarray Analysis; Microdissection; RNA; RNA, Neoplasm; Staining and Labeling; Testicular Neoplasms; Testis

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

U2 - 10.1371/journal.pone.0005536

DO - 10.1371/journal.pone.0005536

M3 - Journal article

C2 - 19436754

SN - 1932-6203

VL - 4

SP - e5536

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 5

ER -