Combining RNAscope and immunohistochemistry to visualize inflammatory gene products in neurons and microglia

A challenge for central nervous system (CNS) tissue analysis in neuroscience research has been the difficulty to codetect and colocalize gene protein expression same tissue. Given importance of identifying relative proteins interest, example, cell-type specific markers, we aimed develop a protocol optimize their codetection. RNAscope fluorescent situ hybridization (FISH) combined with immunohistochemistry (IHC) fixed sections allows reliable quantification transcripts interest within IHC-labeled cells. This paper describes new method simultaneous visualization FISH IHC thicker (14-μm), samples, using spinal cord sections. method’s effectiveness is shown by cell-type-specific two genes, namely proinflammatory cytokine interleukin-1beta (IL-1b) inflammasome NLR family pyrin domain containing 3 (NLRP3). These genes are challenging measure accurately due nonspecificity available antibodies hard-to-distinguish, dot-like visualizations labeled measurements were carried out after unilateral chronic constriction injury sciatic nerve induce neuroinflammation cord. used label antigens (IBA1 microglia, NeuN neurons). combination allowed RNA be quantified boundaries confocal microscopy standard image methods. makes it easy answer empirical questions that intractable or alone. The method, which optimized minimize preparation time costs, described detail from collection analysis. Further, changes inflammatory NLRP3 IL-1b microglia vs. neurons somatotopically relevant laminae first time.