Supplementary Figure 3: CLEM and CLSM to identify Lewy pathology.
From: Lewy pathology in Parkinson’s disease consists of crowded organelles and lipid membranes
(a-f) Light microscopy (LM) image and correlating electron microscopy image (EM) for aSyn inclusions. 150 nm tissue sections collected on LM slides were processed using the LB509 antibody and immunopositive aggregates identified using a peroxidase detection system and green chromogen. Slides were counterstained with hematoxylin in order to identify cellular features for correlation with EM images. The same immunopositive inclusion is indicated (dashed green circle) in an adjacent 150 nm tissue section collected on an EM grid. White arrows indicate tissue features that were used for correlating the LM and EM images. (a) CLEM for Fig. 1d and Supplementary Fig. 8, Donor C-PD, (b-d) CLEM for Supplementary Fig. 6d-f, respectively, Donor D-PDD, (e-f) CLEM for Supplementary Fig. 10 and Fig 6, respectively, Donor E-PD. (g, h) Examples of differential antibody staining for two aSyn-immunopositive inclusions. Adjacent tissue sections were stained with either LB509, phosphorylated aSyn (pSyn; 11a5) antibody or ubiquitin (Ubq). The correlating EM picture for each inclusion is shown. (g) CLEM for Supplementary Fig. 9, Donor D-PDD, (h) CLEM for Fig. 5, Donor D-PDD. All scale bars, a-h = 10 µm. (i) CLSM images from a LB in a neuromelanin-containing neuron in the SN of Donor A-PDD, immunolabeled for alpha-synuclein (LB-509), Serine 129 phosphorylated alpha-synuclein (11A5) and ubiquitin (Ubq).
