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R. S. Ashby, F. Schaeffel, M. Feldkaemper; Laser Capture Microdissection of Retinal Glucagonergic Amacrine Cells From Immunostained Frozen Sections for mRNA Analysis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3592. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glucagonergic amacrine cells have been implicated in the modulation of ocular growth in the chicken. The present study aimed to further elucidate the role of these retinal cells in the regulation of eye growth by developing a methodology for the analysis of changes in gene expression within these specific cells, during periods of altered ocular growth, using laser-capture microdissection (LCM) for later microarray analysis.
Eyes of 8 day old chicks were sagitally sectioned with the posterior eye cup immediately frozen in OCT media. Frozen sections were incubated with primary antibodies for 10 min (anti-ZENK rabbit polycolonal 1:400, anti-glucagon mouse polyclonal 1:40), before being incubated with secondary antibodies for a further 10 min (Oregon Green goat anti-mouse 1:80, Cy3 labeled goat anti-rabbit 1:200). Sections were then rapidly dehydrated through an alcohol series and cleared in xylene. Immunolabeled cells were immediately microdissected using a PALM LCM system (Zeiss). Total RNA was isolated using an RNeasy micro isolation kit (Qiagen). RNA quality and quantity were tested using a bioanalyzer (Agilent) and NanoDrop (Peqlabs) system. ZENK, glucagon, 18S and β-actin mRNA levels were measured using real time-PCR.
Cell specific immunolabeling for ZENK and glucagon could be clearly seen using the rapid staining protocol for LCM, with the results comparable to those seen in 4% paraformaldehyde fixed sections stained using normal immunofluorescent protocols. This allowed for clear identification of ZENK positive glucagonergic amacrine cells from chick retina using LCM. Analysis of samples indicated that roughly 50 ng of total RNA could be isolated per 100 cells. The expression of ZENK, glucagon, 18S and β-actin mRNA was detected using RT-PCR, indicating that ZENK positive glucagonergic amacrine cells had indeed been isolated.
We successfully used immunofluorescent labeling coupled with LCM to isolate and extract ZENK positive glucagonergic amacrine cells from the chick retina. The high quality RNA obtained from these samples should allow future analysis of specific gene changes within the glucagonergic amacrine cells, during periods of altered ocular growth induced by lenses, using microarray technology.
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