Description
Spatial transcriptomics has become an essential tool in retinal research, yet preservation of both tissue architecture and RNA integrity remains technically challenging for post-mortem human samples. Difficulties in working with enucleated globes include retinal detachment from retinal pigment epithelium layer and retinal folding, which commonly occur during dissection due to the retina’s thin and fragile laminated structure. This becomes particularly challenging when seeking to isolate defined regions of interest such as the macula. We have developed a sucrose-gelatin–based stabilisation and embedding protocol that minimizes retinal distortion, followed by cryopreservation of the entire globe prior to regional dissection. Using methylene blue staining, hematoxylin and eosin (H&E) staining, and immunohistochemistry, we demonstrate preservation of macular architecture and retinal lamination. Fluorescent staining for RNA quality from processed samples suggest compatibility with RNA-targeted probe hybridisation, supporting applicability to downstream spatial transcriptomics workflows. This protocol provides a practical solution for spatial analyses of enucleated globes including human tissue and may facilitate similar studies in other mammalian specimens.
Lay Abstract
Spatial transcriptomics is a powerful technique that allows scientists to map which genes are active in different cell types within the retina. However, working with human donor eyes is difficult because the retina is extremely delicate and can easily become damaged or detached during sample preparation.
We developed a new, practical method to keep the eye intact. Our technique uses a specialised mixture of sugar (sucrose) and gelatin to support the eye, similar to filling a fragile ornament with a protective cushion. This mixture stabilises the tissue during freezing and preparation for microscopic study. It also reduces distortion during cryosectioning and when selecting regions of interest from each sample.
We tested the method using a range of staining techniques that show tissue structure and cellular organisation. These confirmed that key features of the retina, including the layered architecture of the macula, were well preserved. We also found that the preserved tissue is suitable for detecting RNA, suggesting compatibility with downstream spatial transcriptomics methods. This protocol provides a reliable way to map protein and gene expression. Using this approach, we can study the human eye in high detail without losing important spatial information between different cell types.
| Lay Title | Keeping Retinal Tissue Intact for Better Spatial Analysis |
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| Role | Postdoctoral Researcher |