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Updated: Dec 27, 2025
ACT-PRESTO: Biological Tissue Clearing and Immunolabeling Methods for Volume Imaging
Published on: December 31, 2016
Lossless immunocytochemistry using photo-polymerized hydrogel thin-films
Jeong Hyun Lee1, Aline T Santoso, Emily S Park
1Department of Mechanical Engineering, University of British Columbia, Vancouver, BC, Canada. hongma@mech.ubc.ca.
Insights
This study introduces a novel hydrogel encapsulation method to prevent cell loss during immunocytochemistry (ICC), ensuring accurate phenotyping for rare cell research. This lossless ICC technique enhances diagnostic applications by preserving cell integrity.
Area of Science:
- Cell Biology
- Biotechnology
- Microscopy
Background:
- Immunocytochemistry (ICC) is crucial for cell phenotyping in research and diagnostics.
- Standard ICC methods suffer significant cell loss (<10,000 cells), impacting rare cell analysis (e.g., circulating tumor cells).
- Existing protocols are time-consuming and incompatible with automation due to multiple centrifugation steps.
Purpose of the Study:
- To develop a lossless ICC protocol for small cell samples.
- To improve the efficiency and automation compatibility of ICC.
- To eliminate cell loss during ICC procedures.
Main Methods:
- Encapsulation of cell samples in a photo-polymerized hydrogel thin-film.
- Utilizing hydrogel permeable to ICC reagents for standard protocol compatibility.
- Physically constraining cells within the hydrogel on microtiter plates.
Main Results:
- Standard ICC and Cytospin protocols exhibit >70% cell loss with <10,000 cells.
- Hydrogel encapsulation results in a lossless ICC process.
- The method requires only a single centrifugation step, reducing protocol time.
Conclusions:
- Hydrogel encapsulation offers a lossless ICC solution for small and rare cell samples.
- This technique enhances ICC reliability for research and diagnostics.
- The method is time-efficient and compatible with robotic platforms.
Abstract:
Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological technique for phenotyping cells in both research and diagnostic applications. Standard ICC methods often do not work well when the cell sample contains a small number of cells (<10 000) because of the significant cell loss that occurs during washing, staining, and centrifugation steps. Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, where such losses could significantly bias experimental outcomes. In order to eliminate cell loss in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, thereby allowing the use of standard ICC protocols without modification. The cell sample is physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several centrifugation steps during staining and washing, our hydrogel encapsulation method requires only a single centrifugation step. This property greatly reduces the time required to perform ICC protocols and is more compatible with robotic platforms. In this study, we show that standard ICC and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10 000 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC process.
