Fig. 2: IMC analysis of mitochondrial MQC proteins expression in human SN neurons.

a An outline of the IMC workflow on FFPE midbrain sections. Immunostaining of metal conjugated antibodies (Table 1) for IMC detection were performed in two experiments, using EDTA (pH = 8) and sodium citrate (pH = 6) buffer respectively for heat-mediated antigen retrieval. Individual dopaminergic neurons within the SN were segmented based on positive tyrosine hydroxylase (TH) staining, a clear nucleus (anti-Histone H3) and intracellular neuromelanin signals (visualised via Ir-intercalator binding); single pixel intensities were extracted, allowing measurement of the mean pixel intensity of each cytoplasmic area for statistical analysis. These neurons were further categorised into those with complex I/IV/V deficiency and those with normal protein levels respectively; the level of OxPhos protein deficiency was statistically defined based on the lower 10% limit of the prediction interval of the control dataset²³. Differences of the signalling protein expression between the two categories (deficient/normal) of neurons were further analysed to understand their changes in association with OxPhos deficiency. Detailed information of the tissue cohort included in the study were summarised in Supplementary Table 1. Scale bar, 20 µm. Example IMC images were selected from PD08 (male, 90 yrs), POLG01 (female, 90 yrs) and CON04 (female, 54 yrs) (b); PD01(male, 70 yrs), POLG02 (male, 59 yrs) and CON09 (male, 88 yrs) (c), demonstrating differential signal intensities from test proteins between neurons within selected individuals, and between groups. Scale bar, 20 µm.