BACKGROUND: Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser. METHODS: To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intravenously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 microm thin polyethylene membrane. Ten glomeruli were collected from 6 microm frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR). RESULTS: Transforming growth factor-beta1 (TGF-beta1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-beta1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6). CONCLUSIONS: We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-beta1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.
BACKGROUND: Laser-manipulated microdissection (LMM) is a method to cut out a single cell or limited tiny region from a specimen under microscopic observation by a laser beam. Laser pressure catapulting (LPC) is a method to push up and collect samples that were microdissected using a strong laser. METHODS: To induce experimental glomerulonephritis, anti-Thy1.1 monoclonal antibody (OX-7) was injected intravenously into rats. Control and disease model kidneys were obtained. Six-micrometer thick cryostat sections were mounted onto a 1.35 microm thin polyethylene membrane. Ten glomeruli were collected from 6 microm frozen sections of rat kidney by LMM and LPC. Isolated glomeruli were used to quantitate the expression of mRNA by real-time polymerase chain reaction (PCR). RESULTS:Transforming growth factor-beta1 (TGF-beta1) mRNA was not detected in glomeruli isolated by the LMM and the LPC methods on day 0, although G3PDH mRNA was measurable in the same samples. On day 7 after the treatment with OX-7, the ratio of TGF-beta1/G3PDH mRNA was 1.89 +/- 0.96 (N = 6). CONCLUSIONS: We established methods to isolate glomeruli from standard histochemical specimens by LMM and LPC, and to quantify mRNA expression in the targeted glomeruli using real-time PCR. We confirmed the up-regulation of TGF-beta1 mRNA expression in isolated glomeruli from frozen sections of the anti-Thy1.1 glomerulonephritis model.
Authors: Xin Shi; Jörg Kleeff; Zhao-Wen Zhu; Bruno Schmied; Wen-Hao Tang; Arthur Zimmermann; Markus W Buchler; Helmut Friess Journal: World J Gastroenterol Date: 2003-06 Impact factor: 5.742
Authors: M Yanagita; H Arai; K Ishii; T Nakano; K Ohashi; K Mizuno; B Varnum; A Fukatsu; T Doi; T Kita Journal: Am J Pathol Date: 2001-04 Impact factor: 4.307
Authors: Clemens L Bockmeyer; Juliane Wittig; Karen Säuberlich; Philipp Selhausen; Marc Eßer; Philip Zeuschner; Friedrich Modde; Kerstin Amann; Christoph Daniel Journal: BMC Mol Biol Date: 2018-03-13 Impact factor: 2.946