Turn your IHC slides into quantitative data.

An important advantage of immunohistochemistry (IHC) is that it maintains the histological structure of a tissue. This allows for the measurement of protein expression in different parts of the tissue down to the cellular and even subcellular level.

Quantifying IHC in each compartment allows one to get additional insight into the biology of the tumor. Epithelial Cell adhesion and Activating Molecule (EpCAM) is a tumor-associated antigen that identifies cells of epithelial origin [1]. EpCAM-based tumor classification is particularly useful when: (a) target marker quantification is required in tumor and stromal regions (b) target marker is absent in tumor cells but present in stromal compartment; (c) target marker is expressed only in tumor cells and background staining is seen in the stroma.

Staining serial sections with EpCAM and a marker of interest allows us to use the HALO® software from Indica Labs to quantify the IHC staining in the tumor compartment and the surrounding stroma independently. 

EpCAM Staining to Identify Tumor Compartment 

The software is trained to recognize EpCAM positive staining by manually identifying regions that are positive, negative or contain no tissue (glass) as shown by red, green and yellow arrows, respectively, in Figure 1B. Based on the experimenter’s input (Figure 1B), the algorithm is then able to classify the entire image into three categories: EpCAM positive, EpCAM negative and glass as shown in Figure 1C. 

Figure 1: EpCAM Staining to Identify the Tumor Compartment

Quantification of Target Marker using EpCAM Staining 

We apply a series of steps to overlay the classified regions from EpCAM staining onto target markers (Marker B in Figure 2) using serial sections that were stained for EpCAM and Marker B. The following steps outline the entire process.

Figure 2: Quantification of Marker B by result from EpCAM Staining 

Step 1: Create a classifier based on EpCAM positivity as defined in EpCAM Staining. 

  • Red: Tumor
  • Green: Strom
  • Yellow: Glass

Step 2: Create an annotation (red outline) for the tumor compartment based on the EpCAM classifier.

Step 3: Copy annotation onto Marker B image.

Step 4: Analyze Marker B IHC only in the tumor annotation.

This approach allows for the quantification of markers that are differently modulated in the tumor versus the stromal compartment. This is another example of how we at Ensigna Biosystems use cutting edge methods to help our clients answer scientific questions relevant to their drug discovery projects.


  1. Okegawa T, Pong RC, Li Y, Hsieh JT. The role of cell adhesion molecule in cancer progression and its application in cancer therapy. Acta Biochim Pol. 2004;51(2):445-457.