Quantitative In Situ Hybridization for Peptide mRNAs in Mouse Brain

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The objective was to determine the feasibility of using a radioactive capture method (Fuji FLA 2000) and image analysis system for the measurement of peptide mRNA levels in specific brain regions in mice. As a test mRNA, we chose vasopressin (VP) and oxytocin (OT) because they are expressed in abundance in the hypothalamic paraventricular (PVN) and supraoptic nuclei (SON). A comparison was made between free-floating and slide-mounted sections to determine which method yielded better results. Mouse brains were fixed in 4% paraformaldehyde (PFA) and processed for in situ hybridization using 35S-oligonucleotide probes for VP and OT. After overnight hybridization and high stringency washes, 25-μm brain sections and 14C standards were exposed to a BAS-IIIs Fuji imaging film over a range of times (4 h–6 days). Results showed that there was an intense hybridization reaction in the PVN and SON, making it possible to distinguish the specific brain regions. Using Image Gauge Software, the signal was quantified in PVN and SON. A comparison of the different exposure times showed that the signal could be measured after as little as 4 h. The intensity readings increased over time while the calculated radioactivity remained constant. The free-floating method was superior to the slide-based system, providing a lower background and a higher signal. The data illustrates the applicability of the phosphor imaging system for the reproducible measurement of mRNA levels in discrete regions of the mouse brain.



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