June 2023
Volume 64, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2023
Highly multiplexed immunohistochemistry of human retina using the IBEX technique
Author Affiliations & Notes
  • Yuan Tian
    University College London Institute of Ophthalmology, London, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Gregory Patient
    University College London Institute of Ophthalmology, London, London, United Kingdom
  • Yuxuan Meng
    University College London Institute of Ophthalmology, London, London, United Kingdom
  • Bruno Charbit
    University College London Institute of Ophthalmology, London, London, United Kingdom
  • Lorna Fowler
    University College London Institute of Ophthalmology, London, London, United Kingdom
  • Colin Chu
    University College London Institute of Ophthalmology, London, London, United Kingdom
    Moorfields Eye Hospital NHS Foundation Trust, London, London, United Kingdom
  • Footnotes
    Commercial Relationships   Yuan Tian None; Gregory Patient None; Yuxuan Meng None; Bruno Charbit None; Lorna Fowler None; Colin Chu None
  • Footnotes
    Support  Moorfields eye charity
Investigative Ophthalmology & Visual Science June 2023, Vol.64, 5023. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Yuan Tian, Gregory Patient, Yuxuan Meng, Bruno Charbit, Lorna Fowler, Colin Chu; Highly multiplexed immunohistochemistry of human retina using the IBEX technique. Invest. Ophthalmol. Vis. Sci. 2023;64(8):5023.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : A wider appreciation for the importance of understanding the spatial biology of ocular tissues now exists. While significant focus is on transcriptomics, defining cells and processes at a protein level will be complementary and critical. Commercial approaches exist but are not designed for the distinct markers required for ocular tissues. In previous work, we developed the iterative bleaching extends multiplexity (IBEX) method which is open source and can be readily customized to achieve highly multiplexed immunohistochemistry on the same tissue section.

Methods : The retina was dissected from three healthy post-mortem donors obtained via Moorfields Biobank (Ethics reference: 20/SW/0031-2022ETR84). Cryosections underwent cyclic immunohistochemistry, fluorescent confocal microscopy and bleaching using the IBEX technique. Following registration, image analysis and segmentation were performed using Imaris and Aivia software.

Results : We demonstrate an optimized panel of antibodies widely available from common commercial sources and our approach for custom conjugation of ocular-specific antibodies that can be used as a resource for others in the field wanting to employ these approaches. We demonstrate using the retina that a 30-plex panel can be readily achieved (subset shown in Figure 1) on the same section. This outlines most key cellular populations and structures, including markers such as Vimentin, Iba1, HuC/D, Calbindin, Desmin, Chx10, CD44, PKCa, M/L-opsin, S-Opsin, B3-tubulin and IB4. Each antibody marker was morphologically validated for cell position and morphology before cell densities, and contact numbers were measured using Aivia software.

Conclusions : Generating a high-content imaging dataset with IBEX allowed the imaging of almost all key cell types within human retina. While the size of the panel will not isolate every cellular subset identified by RNA sequencing, the open-source and optimized panel presented will facilitate wider access to many research groups in the field to enable a deeper assessment of a range of human retinal diseases than previously possible.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Human retina IBEX

Human retina IBEX

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×