Combined RNAscope and immunohistochemistry staining on duodenal paraffin sections as a new tool to reveal cytolytic potential of intraepithelial lymphocytes.
Technical note (RNAscope combined with IHC) method development
Introduction
Marano et al., describe a method to combine RNAscope with immunohistochemistry to achieve double labelling of perforin transcripts within CD8+ or TcRγδ intraepithelial lymphocytes in small intestine sections of celiac disease patients.
One aspect of celiac disease is an increased intraepithelial lymphocyte count. Two distinct populations of cells are noted to be elevated with celiac disease – CD8+ and TcRγδ+ intraepithelial lymphocytes. Depending upon the stage of the disease, both intraepithelial lymphocyte populations may exert cytolytic properties often resulting in intestinal tissue destruction via molecules such as perforin (a pore-forming protein housed in the secretory granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells) and granzyme (serine proteases released by cytoplasmic granules within cytotoxic T cells and natural killer (NK) cells). To help investigate the cytolytic potential of the 2 distinct cellular subsets, a dual labelling method was developed to visualise perforin expression in both CD8+ and TcRγδ+ intraepithelial lymphocytes. Combining RNAscope and IHC techniques avoids potential cross-reactivity when using 2 antibodies raised in the same species plus other potential multi-antibody issues.
Main Points
- RNAscope is an in situ hybridization technique in which probes are designed to selectively target mRNA. The assay can overcome sensitivity and specificity issues that can arise with routine IHC. RNAscope allows the detection of single mRNA transcripts of a target gene using a double z strategy that allows amplification of the target signal with no background. It may be a useful alternative where suitable IHC reagents are not available for specific biomarkers.
- IHC is a technique for the visualisation of surface and cytoplasmic antigens in cells or tissue sections using antigen specific antibodies. The binding of the antibody to an antigen can typically be detected with an enzymic reaction such as HRP or AP plus a chromogenic substrate. Multiplexing with classical IHC alone is possible but requires compatibility of antigen retrieval methods and sourcing antibodies raised in different species that will not cross-react resulting in non-specific labelling and background.
- Prior to running the dual labelling method – optimised protocols for both the perforin RNAscope and the CD8+ and TcRγδ+ were developed and optimised as single marker protocols.
- Duodenal biopsies were collected from 8 patients with confirmed celiac disease. These samples were fixed in 10% formalin for 24hrs and were processed and sectioned as per routine histology.
- The dual staining of the tissue was performed over 3 days. Day 1: Sectioning, slide drying, endogenous peroxide blocking and antigen retrieval (using retrieval reagents from the RNAscope kit). Antigen retrieval performed once at this step was suitable and adequate for both the RNAscope and the IHC steps.
- Day 2: Further exposure of antigenic sites via a protease plus treatment, RNAscope probe incubation, RNAscope amplification steps (with an increase in amplification step 5 from 30 mins to 1hr), RNA signal detection (incubated with RED alk-phos working solution for better contrast with brown / black IHC stained cells), Blocking to avoid non-specific IHC binding, Incubation with the primary antibody (either the anti CD8+ or anti TcRγδ+ mouse primaries)
- Day 3: Application of IHC secondary antibody (anti-mouse for both as both mouse primaries), DAB detection of the IHC labelling (nickel solution was added to adjust the typical brown signal to a grey/black to allow better contrast with the red of the RNAscope perforin labelling).
- For perforin positive tissues the resultant staining showed black, round cells (CD8+ or TcRγδ+ cells depending on the primary antibody) with small red dots within the cytoplasm demonstrating successful dual labelling within the same cell.
- This technique will allow the researchers to further study the cytolytic properties of distinct intraepithelial lymphocyte subsets which are key cels in the pathogenesis of celiac disease.
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