Our Multiplex Immunolabeling Approach

Immunohistochemistry is routinely used to localize and study the distribution of proteins in tissues. Most staining methods are limited to localizing one or two proteins in samples. With the recent advances in the field of tumor immunology and cancer immunotherapy, there is a growing need to develop multiplex imaging methods that can simultaneously label multiple tumor and immune cell proteins. This provides users valuable information about the complex tumor microenvironment. Localizing more than two proteins is challenging as most commercially available antibodies are either raised in mouse or rabbit, as the cost of generation of antibodies in other species would be expensive. Newer approaches involving barcoded antibodies and DNA amplification require specialized expertise, expensive reagents and equipment that can make the process cumbersome.

At Visikol we have developed methods to visualize up to 20 biomarkers in a single sample. Our approach entails multiple rounds of immunolabeling, followed by imaging and stripping of antibodies between rounds. During each round of immunolabeling, the sample is labeled with three to four antibodies using a combination of directly conjugated and unconjugated antibodies. When conjugated and unconjugated antibodies from the same species are used, double labeling is achieved by sequential labeling.

The sample is first incubated with the unconjugated primary antibody and this is followed by incubation with fluorescently conjugated secondary antibody. The next step involves blocking the free antigen binding sites on the secondary antibody with normal serum from the same species as the primary antibody. Finally, the sample is incubated with fluorescently conjugated primary antibody. After the first round of labeling, the sample is imaged on an Aperio Versa 8 slide scanner from Leica.

We have spent considerable effort developing an effective reagent to strip labelled antibodies from samples. Our stripping reagent which we refer to as EasyPlex is gentle and does not result in morphological changes after the rounds of stripping. It has also been tested on a variety of tissues and target proteins, without a loss of antigenicity.

After the entire set of images are acquired, they are co-registered and are uploaded to BitSlide, our cloud-based sharing platform where clients can visualize and annotate images.


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