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(American Journal of Pathology. 2000;157:1459-1466.)
© 2000 American Society for Investigative Pathology

Technical Advances

Immunostaining for Cell Picking and Real-Time mRNA Quantitation

Ludger Fink^*, Thomas Kinfe^*, Werner Seeger, Leander Ermert^*, Wolfgang Kummer and Rainer Maria Bohle^*

From the Departments of Pathology^*
and Internal Medicine,
and the Institute for Anatomy and Cell Biology,
Justus-Liebig-University, Giessen, Germany

Microdissection techniques allow a cell-type or even cell-specific mRNA analysis within complex tissues. Furthermore, valid mRNA quantitation can be performed by real-time reverse transcriptase-polymerase chain reaction from a few isolated cells obtained from cryosections. For a more precise access to many cell types, this technique has to be complemented by a cell-type-specific immunostaining. To evaluate its effect on mRNA quantitation, we analyzed alveolar macrophages (AMs) from control rat lungs and those undergoing stimulation with lipopolysaccharide and interferon- nebulization. Whereas AMs from the left lung were directly harvested for mRNA extraction by bronchoalveolar lavage, tissue sections of the right lung were stained with an optimized immunofluorescence protocol detecting AMs. Fifteen AM profiles per sample were picked by laser-assisted sampling technique. Normalizing to a standard gene, nitric oxide synthase II (NOSII) and tumor necrosis factor (TNF)- mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. In stimulated lungs, the percentage of picked samples positive for NOSII or TNF- mRNA increased significantly. Moreover, a marked increase in the ratio of target gene mRNA to standard gene mRNA was noted for both NOSII and TNF- in picked AMs from stimulated lungs, which matched very well the increase detected in the lavaged AMs undergoing direct RNA extraction. Thus, when using an optimized protocol for immunofluorescence, this approach may be reliably combined with laser-assisted cell picking and real-time mRNA quantitation in a few immunohistochemically characterized cell profiles within complex tissues.

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