A quantitative in situ hybridization protocol for formalin-fixed paraffin-embedded archival post-mortem human brain tissue.
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=0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus.
Liu
Chun-Qing
CQ
Department of Neurobiology, Institute of Neuroscience, Zhejiang University School of Medicine, Hangzhou, China.
Shan
Ling
L
Balesar
Rawien
R
Luchetti
Sabina
S
Van Heerikhuize
Joop J
JJ
Luo
Jian-Hong
JH
Swaab
Dick F
DF
Bao
Ai-Min
AM
D000368:Aged; D000369:Aged, 80 and over; D001344:Autopsy; D001345:Autoradiography; D001923:Brain Chemistry; D003346:Corticotropin-Releasing Hormone / Q000032:analysis; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D006640:Histidine Decarboxylase / Q000032:analysis*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D008875:Middle Aged; D016612:Paraffin Embedding; D010286:Paraventricular Hypothalamic Nucleus / Q000737:chemistry; D010300:Parkinson Disease / Q000201:enzymology*; D013462:Sulfur Radioisotopes; D016707:Tissue Fixation
D005404:Fixatives; D013462:Sulfur Radioisotopes; D005557:Formaldehyde; D003346:Corticotropin-Releasing Hormone; D006640:Histidine Decarboxylase