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