H Murakami1, L Liotta, R A Star. 1. Renal Diagnostics and Therapeutics Unit, and Laboratory of Pathology, National Institutes of Health, Bethesda, Maryland 20892-1268, USA.
Abstract
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 reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression. 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 minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary 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 microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase. CONCLUSIONS: We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.
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 reaction (RT-PCR) can measure gene expression in normal, cancerous, injured, or fibrotic tissue. Nevertheless, targeting of specific cells may be difficult using routine morphologic stains. Immunohistochemistry can identify cells with specific antigens; however, exposure to aqueous solutions destroys 99% of the mRNA. Consequently, there is an overwhelming need to identify specific tissue cells for LCM without mRNA loss. We report on a rapid immunofluorescent LCM (IF-LCM) procedure that allows targeted analysis of gene expression. 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 minute incubation with anti-Tamm-Horsfall antibody and an ALEXA-linked secondary 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 microdissected without contamination from surrounding tubules. The recovery of RNA following rapid immunofluorescence staining was similar to that obtained following hematoxylin and eosin staining, as assessed by RT-PCR for malate dehydrogenase. CONCLUSIONS: We conclude that the new apparatus and method for the immunofluorescent labeling of tissue cells targeted for LCM can isolate pure populations of targeted cells from a sea of surrounding cells with highly acceptable preservation of mRNA. Since the TAL is minimally injured following ischemia, identification of the different responses between TAL and surrounding tissue in damaged kidneys may provide new therapeutic targets or agents for the treatment of acute renal failure.
Authors: Daria Barwinska; Michael J Ferkowicz; Ying-Hua Cheng; Seth Winfree; Kenneth W Dunn; Katherine J Kelly; Timothy A Sutton; Brad H Rovin; Samir V Parikh; Carrie L Phillips; Pierre C Dagher; Tarek M El-Achkar; Michael T Eadon Journal: J Vis Exp Date: 2020-06-09 Impact factor: 1.355
Authors: Robert M Anthony; Joseph F Urban; Farhang Alem; Hossein A Hamed; Cristina T Rozo; Jean-Luc Boucher; Nico Van Rooijen; William C Gause Journal: Nat Med Date: 2006-07-30 Impact factor: 53.440