Enhanced detection of distinct honeycomb-structured neuronal SMARCC2 cytobodies in Parkinson’s Disease via Cyclic Heat-Induced Epitope Retrieval (CHIER)

Authors:

Amber Carmichael-Lowe, Brionne Fleming, Kreesan Reddy, James Wiseman, Eden Paige Yin, Clinton P. Turner, Richard L. M. Faull, Maurice A. Curtis, Mike Dragunow, Birger Victor Dieriks

Keywords:

CHIER; Cyclic Heat-Induced Epitope Retrieval; chromatin-binding proteins; aggregated proteins; neurodegenerative disease; SMARCC2; Parkinson’s Disease

Abstract:

Antigen retrieval is crucial for immunohistochemistry, particularly in formalin-fixed paraffin-embedded brain tissue, where fixation causes extensive crosslinking that masks epitopes. Heat Induced Epitope Retrieval (HIER) reverses these crosslinks, improving access to nuclear and aggregated proteins. We introduce Cyclic Heat-Induced Epitope Retrieval (CHIER), an advanced technique that builds on HIER by incorporating repeated cycles of heating and cooling. CHIER optimises antigen retrieval and significantly improves detection. CHIER is particularly effective for detecting chromatin-binding proteins, such as SMARCC2, which are difficult to label using conventional IHC methods. Using CHIER on formalin-fixed paraffin-embedded human brain sections, we achieved robust detection of SMARCC2 in both the nucleus and cytoplasm. CHIER also enhanced the visualisation of large SMARCC2+ cytoplasmic bodies, termed cytobodies, which are increased in Parkinson’s Disease (PD). Our findings suggest that SMARCC2 may translocate from the nucleus to the cytoplasm in PD, potentially implicating SMARCC2 aggregation in the disease’s pathology. Furthermore, CHIER does not negatively impact the antigenicity of other antibodies, supporting its use for multiplex fluorescent immunohistochemistry and super-resolution imaging. These results highlight CHIER’s potential for improving the detection of chromatin-binding and aggregated proteins in neurodegenerative disease research, offering new insights into SMARCC2’s role in Parkinson’s Disease.

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