Alexa fluor 647

To study the effect of the compounds on every biological target in co-cultured THP-1 immune cells with cancer cell line HT-29, the compounds were incubated for 24 h/48 h as described before.
To detect membrane CD11b and CD80, after the cell incubation, suspended THP-1 was collected from the cell culture of each sample well, fixed with 4% in PBS paraformaldehyde, and stained with FITC Mouse monoclonal Anti-Human CD80 (Sigma-Aldrich, S. Louis, MO, USA, SAB4700142) and Alexa Fluor^® 647 Rabbit monoclonal Anti-CD11b (Merck, S. Louis, MO, USA, #MABF366).

Differentiated glioblastoma cells were seeded in 24-well plates (Sarstedt, Nümbrecht, Germany) with a density of 1500 cells/well, on glass coverslips (Langenbrinck GmbH, Emmendingen, Germany). Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 20 min. The cells were then permeabilized with 20% Methanol for 15 min. To block unspecific binding of antibody, cells were then incubated with 20% Bovine serum albumin (BSA) at RT for 60 min. Primary antibody mix (200 μL; rabbit polyclonal anti-connexin-43 (Sigma, St. Louis, MO, USA) and rabbit monoclonal ß-tubulin (Cell signaling, Cambridge, UK), dilution 1:1000) was added to each coverslip containing wells and incubated at 37 °C for 60 min. Incubation with secondary antibodies (goat anti-rabbit Alexa Fluor 488 and Alexa Fluor 647 (Sigma, St. Louis, MO, USA), dilution 1:1000) was performed at RT for 60 min. DAPI Fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA) was used for nuclei staining. Images were taken with the use of AX 70 microscope and processed with Zeiss Zen software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Fluorescence intensity was calculated as follows: integrated density—(Area of selected cells × mean fluorescence of background).

The total protein of brain tissue was harvested and quantified with a Beyotime Protein Assay Kit, which was used for the following Western blotting. As for detecting Nrf2 in cell samples, nucleus extract of protein lysate was used. After denaturation, the proteins were separated by electrophoresis on polyacrylamide gels, followed by transfer of the proteins to polyvinylidene difluoride membranes, which were blocked with bovine serum albumin (BSA). Then, the membranes were incubated with primary antibodies, followed by incubation with HRP‐conjugated secondary antibody (Jackson 111‐035‐003). Using an enhanced chemiluminescence plus detection system (Pierce Biotechnology), the protein signals on the membranes were quantified by scanning densitometry with image analysis software (Science Lab 2005 Image Gauge).
As for immunofluorescence (IF) staining, sections of brain tissue with 5‐μm thickness were permeabilized with 0.5% Triton X‐100, followed by blocking with 5% BSA. After primary antibody incubation overnight at 4°C, the sections were incubated with Alexa Fluor 488‐ and Alexa Fluor 647‐conjugated secondary antibodies with DAPI (Sigma) for the nuclei staining. The images were examined with an LSM‐880 confocal microscope (Carl Zeiss).

We included multi-labelling immunofluoresecent staining with axonal and myelin markers to visualize the axonal morphology and cytoskeletal abnormalities in 3D with CSLM. Adjacent 60-μm thick sections from PD(D) and DLB cases were rinsed in TBS, pre-treated with EDTA-Tris (pH 9.0) in a steamer (95 °C) and incubated in a cocktail of the following primary antibodies: (1) mouse  anti-myelin proteolipid protein (PLP, 1:500, MCA839G, plpc1, Bio-Rad, Netherlands) and (2) chicken anti-neurofilament heavy chain (NfH, 1:500, AB5539, heavy-chain, Millipore) diluted in TBS, 2% normal donkey serum, and 0.5% Triton-X. Immunostaining was performed for 48 h at 4 °C followed by incubation with secondary antibody goat anti‐chicken coupled with Alexa Fluor 594 (1:400; Molecular Probes, Waltham, MA), donkey anti‐mouse coupled with Alexa Fluor 647, and diamidino‐2‐phenylindole [4 (link), 6 (link)] dihydrochloride (DAPI; Sigma) for nuclear staining for 2 h in the dark. Tissue samples were subsequently rinsed in TBS and blocked in 5% normal mouse serum for 1 h followed by incubation with Alexa488-conjugated mouse anti-phosphorylated-Serine129 (pSer129) α‐synuclein antibody (1:100, 11A5, gift from Prothena Biosciences Inc., San Francisco, CA) for 2 h at 4 °C in the dark. The tissue sections were then mounted on glass slides and cover‐slipped with mowiol-DABCO as a mounting medium (4‐88 Calbiochem).