Because there is, to our knowledge, no publication of brain nuclei specific differences in methylation pattern, we are unable to definitively ascertain IFAP's sensitivity for detecting methylation differences. Although this method minimizes inclusion of non-nuclei tissue by reducing section thickness requirements, it does remain limited by cell heterogeneity (i.e., neuronal versus glial) in the target nuclei and is unable to discriminate cell-type restricted changes in DNA methylation. Notwithstanding this limitation, the successful extraction of RNA, conversion, and amplification of TPH2 and LMX1b cDNA suggests a level of sensitivity comparable to MP, while offering increased targeting accuracy and streamlined processing when using conventional slide-mounted frozen tissue sections.
In conclusion, IFAP is a flexible, low-cost, and simple method for dissecting nuclei from thin slide-mounted sections. The method synergizes well with conventional histochemical techniques, allowing for parallel investigations with tissue derived from the same animal. The central advantages of IFAP over MP are (i) seamless integration into the histochemical workflow, (ii) quantitative recovery from thin, slide-mounted frozen sections, (iii) no need for specialized equipment, and (iv) targeting accuracy using ISH. We verified that gDNA and RNA extracted by this method can be used in DNA methylation analysis and cDNA synthesis, respectively.
We would like to thank Delia Vazquez, Juan Lopez, Robert Thompson, and Robert Denver for their input. This work was supported by a grant from the Pritzker Neuropsychiatric Disorders Research Consortium, which is supported by the Pritzker Neuropsychiatric Disorders Research Fund L.L.C. and a NARSAD Young Investigator Grant to P.D.P. (N008728).
The authors declare no competing interests.
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