Immuno-LCM: Laser capture microdissection of immunostained frozen sectionsfor mRNA analysis

Microdissection of routinely stained or unstained frozen sections has been used successfully to obtain purified cell populations for the analysis of c ell-specific gene expression patterns in primary tissues with a complex mix ture of cell types. However, the precision and usefulness of microdissectio n is frequently limited by the difficulty to identify different cell types and structures by morphology alone. We therefore developed a rapid immunost aining procedure for frozen sections followed by laser capture microdissect ion (LCM) and RNA extraction, which allows targeted mRNA analysis of immuno phenotypically defined cell populations. After fixation, frozen sections ar e immunostained under RNAse-free conditions using a rapid three-step strept avidin-biotin technique, dehydrated and immediately subjected to LCM. RNA i s extracted from captured tissue, DNAse I treated, and reverse transcribed. Acetone-, methanol-, or ethanol/acetone-fixed sections give excellent immu nostaining after 12 to 25 minutes total processing time. Specificity, preci sion, and speed of microdissection is markedly increased due to improved id entification of desired (or undesired) cell types. The mRNA recovered from immunostained tissue is of high quality. Single-step PCR is able to amplify fragments of more than 600 bp from both housekeeping genes such as beta-ac tin as well as cell-specific messages such as CD4 or CD19, using cDNA deriv ed from less than 500 immunostained, microdissected cells. Immuno-LCM allow s specific mRNA analysis of cell populations isolated according to their im munophenotype or expression of function-related antigens and significantly expands our ability to investigate gene expression in heterogeneous tissues .