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首页> 外文期刊>Brain research. Brain research protocols >Quantitative in situ hybridization for peptide mRNAs in mouse brain.
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Quantitative in situ hybridization for peptide mRNAs in mouse brain.

机译：小鼠脑中肽mRNA的定量原位杂交。

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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-microm 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.
机译：目的是确定使用放射性捕获方法（Fuji FLA 2000）和图像分析系统测量小鼠特定脑区域中肽mRNA水平的可行性。作为测试mRNA，我们选择了血管加压素（VP）和催产素（OT），因为它们在下丘脑室旁室（PVN）和视上核（SON）中大量表达。比较了自由浮动部分和滑动安装部分，以确定哪种方法可获得更好的结果。将小鼠大脑固定在4％多聚甲醛（PFA）中，并使用用于VP和OT的35S-寡核苷酸探针进行原位杂交。过夜杂交和高严格度洗涤后，将25微米的脑切片和14C标准品在一段时间（4小时至6天）内暴露于BAS-IIIs Fuji成像胶片。结果显示，PVN和SON中发生强烈的杂交反应，从而可以区分特定的大脑区域。使用Image Gauge Software，信号以PVN和SON量化。对不同曝光时间的比较表明，仅需4小时即可测量信号。强度读数随时间增加，而计算的放射性保持恒定。自由浮动方法优于基于幻灯片的系统，可提供较低的背景和较高的信号。数据说明了荧光粉成像系统在小鼠大脑离散区域中可重复测量mRNA水平的适用性。

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