A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue

Methods : a Companion to Methods in Enzymology
Chun-Qing LiuAi-Min Bao

Abstract

The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800 mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinson's disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p=0.0...Continue Reading

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Citations

Dec 25, 2010·Nanomedicine·Christine VauthierPatrick Couvreur
Oct 3, 2012·Journal of Affective Disorders·Ling ShanAi-Min Bao
Jan 13, 2015·Trends in Neurosciences·Ling ShanDick F Swaab
Jan 31, 2012·Neurobiology of Aging·Ling ShanDick F Swaab
Jan 20, 2012·Brain Pathology·Daniel J van WamelenDick F Swaab
Nov 26, 2010·Brain Pathology·Daniel J van WamelenDick F Swaab
Aug 30, 2012·Journal of Neuroendocrinology·D J van WamelenD F Swaab
Aug 23, 2012·Experimental Gerontology·Ling ShanAi-Min Bao

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