Department of Psychiatry Washington University School of Medicine St. Louis MO USA.
The Taylor Family Institute for Innovative Psychiatric Research Washington University School of Medicine St. Louis MO USA.
Brain Behav. 2017 May 30;7(7):e00736. doi: 10.1002/brb3.736. eCollection 2017 Jul.
With its preservation of cytoarchitecture and synaptic circuitry, the hippocampal slice preparation has been a critical tool for studying the electrophysiological effects of pharmacological and genetic manipulations. To analyze the maximum number of slices or readouts per dissection, long incubation times postslice preparation are commonly used. We were interested in how slice integrity is affected by incubation postslice preparation.
Hippocampal slices were prepared by three different methods: a chopper, a vibratome, and a rotary slicer. To test slice integrity, we compared glycogen levels and immunohistochemistry of selected proteins in rat hippocampal slices immediately after dissection and following 2 and 4 hr of incubation.
We found that immunoreactivity of the dendritic marker microtubule-associated protein 2 (Map2) drastically decreased during this incubation period, whereas immunoreactivity of the glutamate transporter VGlut1 did not significantly change with incubation time. Astrocytic and microglial cell numbers also did not significantly change with incubation time whereas glycogen levels markedly increased during incubation.
Immunoreactivity of the dendritic marker Map2 quickly decreased after dissection with all the slicing methods. This work highlights a need for caution when using long incubation periods following slice preparation.
海马切片制备因其对细胞结构和突触连接的保存，成为研究药理学和遗传学操作对电生理影响的重要工具。为了分析每块切片或每次切片检测的最大数量，通常会在切片后进行长时间的孵育。我们对切片后孵育对切片完整性的影响很感兴趣。
我们通过三种不同的方法制备海马切片：组织匀浆器、振动切片机和旋转切片机。为了测试切片的完整性，我们比较了大鼠海马切片在切割后立即以及孵育 2 小时和 4 小时后的糖原水平和选定蛋白质的免疫组化。
我们发现，在这段孵育时间内，树突标记物微管相关蛋白 2（Map2）的免疫反应性急剧下降，而谷氨酸转运体 VGlut1 的免疫反应性随孵育时间的变化不显著。星形胶质细胞和小胶质细胞数量在孵育过程中也没有显著变化，而糖原水平在孵育过程中显著增加。
用所有切片方法切割后，树突标记物 Map2 的免疫反应性迅速下降。这项工作强调了在切片制备后使用长时间孵育时需要谨慎。
