fISHing with immunohistochemistry for housekeeping gene changes in Alzheimer’s disease using an automated quantitative analysis workflow

In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) determine cell type provides cellular context of expression, which cannot achieved gene microarray or polymerase chain reaction. To study and protein on the same section we investigated use RNAscope® combination fluorescent IHC paraffin-embedded human brain tissue. We first developed high-throughput, automated image analysis workflow for quantifying RNA puncta across total population within neurons identified by NeuN+ immunoreactivity. then applied this (TMA) sections middle temporal gyrus (MTG) from neurologically normal Alzheimer's Disease (AD) cases suitability three commonly housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) DNA-directed II subunit RPB1 (POLR2A). Overall, saw significant decrease neuronal UBC AD compared cases. Total results were validated RT-qPCR using fresh frozen 5 conclude technique combined our novel pipeline suitable platform changes diseased anatomical context. Furthermore, suggest not post-mortem