Probes for KIT

Complex tissues such as tumors are comprised of multiple cells types and extracellular matrix. These cells include heterogenous populations of immune cells that infiltrate the tumors. Understanding the composition of these immune infiltrates in the tumor microenvironment (TME) can provide key insights to guide therapeutic intervention and predict treatment response. Thorough understanding of complex tissue dynamics and immune cell characterization requires a multi-omics approach. Simultaneous detection of RNA and protein using in situ hybridization (ISH) and immunohistochemistry/immunofluorescence (IHC/IF) can reveal cellular sources of secreted proteins, identify specific cell types, and visualize the spatial organization of cells within the tissue. However, a sequential workflow of ISH followed by IHC/IF frequently yields suboptimal protein detection because the protease digestion step in the ISH protocol resulting in poor antibody signal. Here we demonstrate a newly developed integrated ISH/IHC workflow that can substantially improve RNA-protein co-detection, enabling the visualization and characterization of tumor immune infiltrates at single-cell resolution with spatial and morphological context. To characterize tumor-infiltrating immune cells in a tumor TMA (tumor microarray), we utilized the RNAscope Multiplex Fluorescence assay in combination with the RNA-Protein Co-detection Kit to detect multiple immune cell populations. Immune cells such as macrophages, T cells and NK cells were detected using specific antibodies against CD68, CD8, CD4 and CD56, respectively. Precise characterization of these immune cells was achieved by using probes against targets such as CCL5, IFNG, GNZB, IL-12, NCR1 etc. that not only help in identifying specific immune cells but also assist in determining their activation states. We identified subsets of T cells such as CD4+ regulatory T cells and CD8+ cytotoxic T lymphocytes. Additionally, we were able to determine the activation states of CD8+ T cells by visualizing the expression of IFNG and GZMB. Furthermore, infiltrating macrophages were identified by detecting the CD68 protein expression while the M1 and M2 subsets were differentiated by detecting the M2-specific target RNA for CD163. Similarly, NK cells were identified by detecting CD56 protein in combination with CCL5 and NCR1 RNA expression. Interestingly, the degree of infiltration of the different immune cell populations varied based on the tumor type. In conclusion, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies - including many previously incompatible antibodies - with RNAscope. This new workflow provides a powerful new approach to identifying and characterizing tumor infiltrating populations of immune cells.

Purpose: Surgical destabilization of the medial meniscus (DMM) is a widely used mouse model of knee osteoarthritis (OA). The cell bodies of primary sensory neurons innervating the knee joints are located in the lumbar dorsal root ganglia (L3-L5 DRG). Analysis of the gene expression profile of L3-L5 DRG after DMM or sham surgery revealed that innate neuro-immune pathways were strongly regulated, especially in the later stages of the model, 8-16 weeks after DMM, when persistent pain is associated with severe joint damage. In depth analysis of the microarray data further showed that a number of genes encoding molecules in the Notch signaling pathway were regulated, mostly in late-stage disease, along with the upregulation of the gene encoding monocyte chemoattractant protein (MCP)-1/C-C motif chemokine ligand 2 (CCL2). CCL2 is a proalgesic mediator that is released upon tolllike receptor (TLR) 2/4 activation, and plays a key role in initiating and maintaining pain in this model. The aim of this study was to investigate Notch signaling in the knee-innervating DRG of mice with experimental knee OA, and determine the effect of Notch signaling activation on TLR2/4-mediated CCL2 synthesis in cultured DRG cells. Methods: DMM or sham surgery was performed in the right knee of 10- week old male C57BL/6 mice. Ipsilateral L4 DRG from mice 26 weeks after DMM or sham surgery were collected and cryosectioned. Expression of the Notch downstream target gene, Hes1, was detected using RNA in situ hybridization (ISH) (RNAscope, Advanced Cell Diagnostics). Quantification of mRNA expression was performed as calculating H-score of each sample according to the 0-4 five-bin scoring system recommended by the manufacturer, based on the number of cells with the same range of number of dots per cell. Active Notch protein was detected via immunofluorescence (IF) staining using an antibody against Notch intracellular domain (NICD), which is only present after g-secretase cleavage of Notch at S3. For in vitro cultures of DRG cells, bilateral L3-L5 DRG were collected from 10-week old male naïve C57BL/6 mice. Following enzymatic digestion, DRG cells were plated on poly-L-lysine and laminin coated glass coverslips, and cultured in F12 medium supplemented with 1x N2 and 0.5% fetal bovine serum. Inhibition of Notch signaling was achieved by (1) g-secretase inhibitor, DAPT; (2) ADAM-17 inhibitor, TAPI-1; or (3) soluble form of the Jag1 peptide (sJag1). On day 4, cells were pre-treated with DAPT (25 mM), TAPI-1 (20 mM), or sJag1 (40 mM) for 1 hour, followed by addition of the TLR2 agonist, Pam3CSK4 (1 mg/ml), or the TLR4 agonist, LPS (1 mg/ ml). Then, RNA was collected 3 hours later for qRT-PCR to quantify Ccl2 mRNA expression, or culture supernatants were collected 24 hours later to measure the CCL2 protein level using Quantikine Mouse CCL2/JE/ MCP-1 Immunoassay kit from R&D Systems, Inc.