(Designed for RNAscope®)
Indica Labs ISH-IHC measures one IHC stain and up to two chromogenic and/or silver-labeled DNA or RNA ISH probes on a cell-by-cell basis. This allows the user to rapidly contextualize the corresponding protein and gene expression profile of every cell across the tissue. HALO ISH-IHC analysis is designed to work with single or dual IHC-ISH assays including RNAscope™ and supports the H-score protocol for RNAscope as recommended by the manufacturer, ACD Bio.
An example of ISH-IHC quantification is shown in the slider above. Here, CD3 IHC is combined with an RNA ISH probe for the same (red) and hematoxylin counterstain. Cells that are negative for CD3 are blue in the markup image, whereas cells with CD3 expression are green. The ISH probe spots are highlighted in various shades of red according to their size within the identified cells. IHC stains can be identified and quantified in any compartment – nucleus, cytoplasm and/or membrane. While we have used a cytoplasmic radius to identify the cell boundaries in this instance, if you are using a membrane IHC marker then you can use this to define the cell boundaries (and thus more accurately identify the cytoplasmic compartment in each cell).
The module reports numerous outputs including, but not limited to: number of IHC positive cells in total and by cell compartment, Average IHC OD and average IHC OD by cell compartment, percentage IHC positive total and by cell compartment, number and percentage of probe 1 and/or probe 2 positive cells, probe 1 and probe 2 copies per cell and area per cell, cell score for probe 1 and probe 2 (0, +1, +2, +3 and +4), total H-score for each probe, total cells and percentage of total cells positive for IHC marker and probe 1 and/or probe 2 (dual and triple positive cells). Histograms are automatically generated representing the frequency distribution of probes per cell [Figure 1] and RNAscope cell scoring [Figure 2] as shown below.
Figure 1 shows a distribution curve of probe number on a cell by cell basis, in this example most cells were shown to have between 0-5 copies per cell.
Figure 2. This bar chart shows the numbers of cells that were categorized as 0, 1+, 2+, 3+ and 4+ respectively. The chart shows that a slim majority of cells were positive for RNA probe expression.