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

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.

Fig 1. SMARCC2-1A3 labelling in the middle temporal gyrus brain tissue (MTG).

SMARCC2-1A3 (green), NeuN (red), nucleus (blue). (A) In controls, we primarily observed a nuclear punctate SMARCC2 staining pattern. (B) In some controls and all PD cases, circular cytoplasmic SMARCC2⁺ bodies, named SMARCC2⁺ cytobodies, are present. (C) Typical nuclear SMARCC2⁺ labelling. (D) Nuclear SMARCC2⁺ labelling with a SMARCC2⁺ cytobody. (E) The same cell as D was imaged using the optimal setting for SMARCC2⁺ cytobody visualisation. (F) No nuclear SMARCC2⁺ labelling with saturated SMARCC2⁺ cytobody (same image setting as in E). All staining shown in Fig 1 was performed using standard antigen retrieval protocol (10 mM tris-EDTA pH 9). Scale bars represent 10 μm.

Fig 2. Quantification of neurons with SMARCC2+ cytobodies using different Antigen retrieval methods.

(A) Antigen retrieval optimisation methods. (B) Comparison Cyclic Heat Induced Epitope Retrieval (CHIER) to standard and formic acid antigen retrieval using PD cases. (C) Quantification of neurons with SMARCC2⁺ cytobodies in MTG tissue (neurologically normal and PD). Correlation of neurons with SMARCC2⁺ cytobodies with (D) age of death, (E) post-mortem delay, (F) brain weight, (G) years of onset, (H) α-Syn pS129 load. (I) Cell count with SMARCC2⁺ cytobodies separated male/female. (C-I) Staining performed with CHIER protocol D. * p <0.05, ** p <0.01.

Fig 3. SMARCC2/Baf170 antibody comparison.

(A) Schematic diagram depicting the amino acid sequence and structural domains of the SMARCC2 protein, including the various isoforms and an antibody-epitope map of the four epitope-specific SMARCC2/Baf170 antibodies used in this study. (B-C) SMARCC-1A3 (yellow) and SMARCC2 (APO6744PU-N, cyan) labels SMARCC2⁺ cytobodies (yellow and cyan arrows) and nuclear puncta (weak labelling for SMARCC2 (APO6744PU-N). (D) SMARCC2 (ab71907, cyan) and Baf170 (sc17838, magenta) only label nuclear puncta, of which some overlap (red and white arrows) with SMARCC2-1A3 puncta (yellow). SMARCC2 (ab71907, cyan) and Baf170 (sc17838, magenta) never label SMARCC2⁺ cytobodies as seen for SMARCC2-1A3 and SMARCC2 (APO6744PU-N). All labelling in this Fig was performed using CHIER protocol D. Scale bars represent 10 μm for B & C and 1 μm for D.

Fig 4. Western blot of human middle temporal gyrus (MTG) brain tissue, pericytes and recombinant human SMARCC2 protein.

(A) Western blotting of the positive control containing full-length SMARCC2 protein (TP303774, Origene) with SMARCC2-1A3 (red) showed a clear band at 150 kDa, and a faint band at 130 kDa, which overlaps with the SMARCC2 (ab71907; green) bands observed at 150 and 130 kDa. A high-exposure image of the 150 kDa region is shown underneath the entire blot. In MTG (H250, H246, PD65, PD71) and pericytes (H239, H243, PD65, PD78), two clear bands at 60 and 25 kDa were observed with SMARCC2-1A3 and a light band at 150 kDa with SMARCC2 (ab71907). (B) Western blotting of MTG with SMARCC2 (AP06744PU-N, green) detected two clear bands at 130 kDa and 25 kDa, with the 25 kDa band overlapping with the 25 kDa SMARCC2-1A3 band (red). A high-exposure image of the 25 kDa region is shown underneath the entire blot.

Fig 5. Super-resolution imaging of neurons with SMARCC2⁺ cytobodies.

(A) single confocal plane image of neurons with SMARCC2⁺ cytobody. (A’) zoom of SMARCC2⁺ cytobody (B) 3D STED render of SMARCC2⁺ cytobody shown in A. (C) 3D render of neuron with SMARCC2⁺ cytobody. (D-E) 3D renders of SMARCC2⁺ cytobodies. (F) SMARCC2⁺ cytobodies not interacting with α-Syn pS129 aggregates. (G) SMARCC2⁺ cytobodies interacting with α-Syn pS129 aggregates (single confocal plane with orthogonal views). (H) P62 is found within SMARCC2⁺ cytobodies (single confocal plane with orthogonal views). Labelling performed using optimised CHIER protocol D. Scale bars represent 10 μm for A & C and 1 μm for A’, D-H.