Development of an in situ Hybridization Histochemistry for Choline Acetyltransferase mRNA with RNA Probes(Cell and Molecular Biology)

概要

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An in situ hybridization histochemistry (ISHH) for choline acetyltransferase (ChAT) in paraffin sections of the central nervous system of the rat was developed using ^<35>S-labeled RNA probes by defining the technical parameters enabling optimal detection of ChAT mRNA. Fixation by perfusion with 4% paraformaldehyde and 0.4% glutaraldehyde provided the most intense signals. Slides for mounting paraffin sections were coated with 1% BSA and fixed with 25% glutaraldehyde. Proteinase K treatment of tissue sections after fixation increased intensity of signals. Decreasing probe length increased intensity of signals; truncation to approximately 75 nucleotides by alkaline hydrolysis provided the best result. Ribonuclease A treatment after hybridization appeared to be essential to reduce nonspecific background signals. Specificity of hybridization signals was confirmed by the selective localization of signals in motoneurons in the spinal cord with an antisense probe and no signal in them with a sense probe. Distribution patterns of perikarya of neurons in the forebrain, cranial nerve motor nuclei and cervical region of the spinal cord revealed by ISHH and immunohistochemistry (IHC) were almost superimposable, giving additional evidence for specificity of the present method. However, intensity of signals for ChAT mRNA detected by ISHH and cellular content of ChAT protein revealed by IHC were not always parallel.
社団法人日本動物学会の論文
1992-04-15