🧮7. Multiplex

The limitation of conventional immunofluorescence (IF) and immunohistochemistry (IHC) is only a small number (1 - 4) of proteins can be simultaneously labelled and visualized in an experiment. This is due to spectral overlap or cross-reactivity with antibodies. Multiplexing offers a power solution by enabling the simultaneous visualization of multiple markers in the same tissue section. This can be extremely valuable in ENS research, where distinct celltypes such as neuronal subtypes or even glial subtypes can be defined by unique co-expression patterns of different proteins.

The strategy with some of the multiplexed methods are to label the tissue with multiple markers, acquire an image, strip the antibodies or inactivate the fluorophores (depending on the method), label with new combination of markers and keep repeating this. During the multiple rounds, the tissue handling, mounting and staining can induce deformations in the tissue. Moreover, it is challenging to precisely image the same area in the same configuration repeatedly. Thus, we need to align the images from each round.

To facilitate this, one approach is to reserve one channel in every round for a single marker such as DAPI/Hoechst that labels all cell nuclei. We can use this as a reference to figure out the degree of distortion in every round and correct it. Instead of DAPI, you could also use another marker that labels all your cells of interest. For example, Chen et al. (2023) used the pan-neuronal marker Hu as a common marker in every imaging round.

GAT provides macros that facilitate image alignment. Once the images are aligned you can use the neuron counting workflow within GAT to automate the downstream analysis of enteric neurons and their subtypes.

Below is an example of an aligned image from a multiplexing round with ~14 markers through 6 rounds of staining.

Tissue: Myenteric wholemount of human colon tissue

Image from Chen et al., 2023

Multiplex image alignment

Multiplex analysis using GAT

Multiplex analysis using QuPath

Reference:

Chen, B. N., Humenick, A., Yew, W. P., Peterson, R. A., Wiklendt, L., Dinning, P. G., Spencer, N. J., Wattchow, D. A., Costa, M., & Brookes, S. J. H. (2023). Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding. Cellular and molecular gastroenterology and hepatology, 16(4), 573–605.