Ab6564

Each heart was incised transversely along the lower edge of the atrium, embedded with paraffin and cut into 5-µm-thick slices. The sections were dewaxed, rehydrated and blocked with 0.1% (v/v) Triton X-100/0.25% bovine serum albumin (BSA), and were subsequently incubated with various primary antibodies, fluorescently-labeled secondary antibodies and 4, 6-diamidino-2-phenylindole (DAPI, Solarbio, D8200). The following primary antibodies were used: rabbit anti-cardiac troponin T (CTNT, Abcam, ab209813, 1:500), and rabbit anti-CD68 (Boster, BA3638, 1:200). CY3-conjugated anti-rabbit secondary antibody (Abcam, ab6939, 1:200, anti-CD68), or CY5-conjugated anti-rabbit secondary antibody (Abcam, ab6564, 1:500, anti-CTNT) was applied following primary antibody incubation. The images were observed under a fluorescence microscope (Nikon, ECLIPSE 80I). Six 40× fields were randomly selected and taken photos to count the average number of positive cells.

T84 cells were grown on Transwell filters (pore size 0.4 μm) and treated with ASP in HBSS supplemented with 15 mM HEPES, pH 7.4. After incubation for 6 hr at 37°C in a 5% CO₂ atmosphere, the monolayer was washed three times in ice-cold phosphate-buffered saline (PBS) and fixed/permeabilized in ice-cold 100% ethanol at −20°C for 20 min. After being blocked with BlockAid Blocking Solution (Thermo Fisher Scientific, Waltham, MA), the samples were incubated with the primary antibodies against nectin-2 (ab135245, Abcam)1:250, afadin (A0349, Sigma-Aldrich)1:500, and E-cadherin (610181, BD Biosciences)1:500. The secondary antibody was Cy5-conjugated goat anti-rabbit antibody (ab6564, Abcam)1:1000 or FITC-conjugated goat anti-mouse antibody (sc-2010, Santa Cruz Biotechnology)1:200.
The samples were then washed three times with PBS and mounted in ProLong Gold mount gel (Thermo Fisher Scientific) with propidium iodide (PI; Nacalai Tesque). Fluorescence signals were visualized with a confocal laser scanning microscope (FV-300, Olympus, Tokyo). All experiments were performed in triplicate, and the reproducibility of the results was confirmed.

Brain sections were incubated in PBS buffered blocking solution containing 2% normal donkey serum and 0.2% Triton X-100 for 1 hour at room temperature, followed by incubation overnight at 4°C in blocking solution containing primary antibodies: anti-alpha-synuclein (1:500, ab80627, abcam); anti-iba1 (1:500, 019–19741, Wako); anti-mCherry (1:1000, PA5-34974, Thermo Fisher); anti-TH (1:500, ab1542, ab152, Sigma-Aldrich), Nissl neurotrace 640/660 (1:300, Thermo Fisher N21483). The sections were washed three times in PBS and then incubated in one of the following secondary antibodies for 1 h in the dark at room temperature: donkey anti-rabbit Alexa Fluor® 488(1:500, A21206, Thermo Fisher); donkey anti-mouse Alexa Fluor® 488 (1:500, A21202, Thermo Fisher); donkey anti-sheep Alexa Fluor® 488 (1:500, A11015, Thermo Fisher); Cy5 ® (1:500, ab6564, Abcam). Sections were washed three times in PBS, mounted onto slides, coverslipped with ProLong® Gold Antifade Reagent with DAPI (8961S, Cell Signaling). For detection of the phosphorylated form of α-syn at serine 129 (pSer129) sections were incubated in formic acid for 5 min before proteinase K digestion (Enzo Life Sciences, Farmingdale, NY) (1:100 for 15 min at 37°C water followed by 5 min at room temperature) prior to incubation with primary antibody pSer129 α-syn (1:500, ab51253, Abcam).

T84 cells were grown on Transwell filters (pore size 0.4 µm) and treated with various bacterial solutions suspended in HBSS containing 15 mM HEPES and pH 7.4 at MOI = 5. After incubation for 3 hr at 37°C in a 5% CO₂ atmosphere, the monolayer was washed three times in ice-cold PBS and fixed/permeabilized in ice-cold 100% ethanol at −20°C for 20 min. After being blocked with BlockAid™ Blocking Solution (Thermo Fisher Scientific), the samples were incubated with the primary antibodies against ZO-1 (610966, BD Biosciences) 1:500 and claudin-7 (ab27487, Abcam) 1:100. The secondary antibody was Cy5-conjugated goat anti-rabbit antibody (ab6564, Abcam) 1:1000 or Alexa Fluor 594-conjugated goat anti-mouse antibody (ab150116, Abcam) 1:1000. Samples were washed with PBS, and then 4’,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) was applied for 10 min to stain the nuclei. After a final wash with PBS, the membranes were transferred to a glass slide and mounted in ProLong™ Gold mount gel (Thermo Fisher Scientific). Fluorescence signals were visualized with a confocal laser scanning microscope (LSM800, Carl Zeiss, Oberkochen, Germany).

Confocal microscopy was performed to detect intracellular GRK2 expression. Mouse blood cells were subjected to cytospinning and were fixed, permeabilized, and stained with an anti-GRK2 antibody (1:100, ab32558, Abcam, Cambridge, UK) as the primary antibody. Goat anti-rabbit IgG H+L Cy5^® (1:400, ab6564, Abcam, Cambridge, UK) was used as the secondary antibody. Stained cells were counterstained with DAPI and analyzed with a FLUOVIEW confocal microscope (FV10i, Olympus America, Melville, NY, USA).