IF-LCM: Laser capture microdissection of immunofluorescently defined cellsfor mRNA analysis Rapid Communication

Background The next phase of the molecular revolution will bring functional genomics down to the level of individual cells in a tissue. Laser capture microdissection (LCM) coupled with reverse transcription-polymerase chain r eaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless. targeting of specific cells may be diffi cult using routine morphologic stains. Immunohistochemistry can identify ce lls with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify s pecific tissue cells for LCM without mRNA loss. We report on a rapid immuno fluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene ex pression. Methods. A LCM microscope was outfitted for epifluorescence and light level video microscopy. Heat filters were added to shield the image intensifier from the laser. Frozen sections were fluorescently labeled by a rapid one m inute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secon dary antibody. Fluorescently labeled thick ascending limb (TAL) cells were detected by low light level video microscopy, captured by LCM, and mRNA was analyzed by RT PCR for basic amino acid transporter, Tamm-Horsfall protein , and aquaporin-2. Results. The immunofluorescently identified TAL could be cleanly microdisse cted without contamination from surrounding tubules. The recovery of RNA fo llowing rapid immunofluorescence staining was similar to that obtained foll owing hematoxylin and eosin staining, as assessed by RT-PCR for malate dehy drogenase. Conclusions. We conclude that the new apparatus and method for the immunofl uorescent labeling of tissue cells targeted for LCM call isolate pure popul ations of targeted cells from a sea of surrounding cells with highly accept able preservation of mRNA. Since the TAL is minimally injured following isc hemia, identification of the different responses between TAL and surroundin g tissue in damaged kidneys may provide new therapeutic targets or agents f or the treatment of acute renal failure.