Abstract
Purpose :
We have previously used Immuno Tomography (IT) to identify label retaining stem cell populations in the cornea and meibomian gland. While this method provides the unique ability to quantify stem cell populations comprised of 1-4 cells, the number of antigens that can be sequentially used to characterize these unique cells is limited by the antigen stability after antibody stripping and re-probing. To address this deficiency, we have evaluated the capability of imaging mass cytometry (IMC) to generate multiplexed images using metal-conjugated antibodies to label IT plastic sections and generate 3-dimensional IMC data sets (3D-IMC).
Methods :
K5-H2B-GFP mice, 56 days after doxycycline chase were sacrificed and eyelid tissue processed for IT. A total of 400 serial, plastic sections, 2 mm thick were then probed using metal tagged antibodies specific for sox9, collagen type I, E-cadherin, Ki67, GFP, aSMA, vimentin and DNA Intercalator. Multiplexed images were then generated using an Imaging Mass Cytometery system (Fluidigm), and 3D reconstructions assembled.
Results :
All 8 metal labeled tags were detected and their images were successfully assembled into 3D-IMC data sets. GFP labeled nuclei were identified within the meibomian glands in comparable numbers to those previously reported for slow cycling, meibomian gland stem cells.
Conclusions :
These findings demonstrate that IMC can be used on plastic sections to generate multiplexed, 3D data sets that can be reconstructed to show the spatial localization of meibomian gland stem cells. We propose 3D-IMC might prove valuable in more fully characterizing stem cell populations in different tissues.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.