Alexa fluor conjugates

For immunofluorescence analysis, paraffin-embedded rat vagus nerve section (5 μm) were dewaxed and rehydrated, then subjected to antigen retrieval using citrate buffer (pH 9) at 95 °C for 45 min. Next, sections were incubated with a blocking solution containing 0.3% Triton X-100 for permeabilization and 3% BSA for 20 min at room temperature, and then incubated with the following primary antibodies at 4 °C overnight: Anti-alpha-synuclein (Abcam ab280377, 1:500), Anti-phospho S129 alpha-synuclein (Abcam ab51253, 1:500), Anti-S100 beta (Abcam ab52642, 1:500), Anti-S100 beta (Proteintech 66616-1-Ig, 1:500), Anti-Neurofilament heavy polypeptide (Abcam ab207176, 1:200), Anti-MJF-14 (Abcam ab209538, 1:500), Anti-TLR2 (Abcam ab209216, 1:500), Anti- interleukin-1 beta (IL-1β) (Santa cruz sc-12742, 1:50). After washing 3 times in PBS, nerve sections were incubated with corresponding secondary antibodies at 1:2000 (Abcam, Alexa Fluor conjugates) for 1 h at room temperature. DAPI (Sigma-Aldrich) was used for staining nuclei. Images were acquired with a confocal laser scanning microscopy (Zeiss LSM700, Oberkochen, Germany).

After being dewaxed and rehydrated, the paraffin-embedded mice vagus nerve section (5 µm) was antigenically repaired using citrate at 100 °C for 50 min. RSC96 cells were seeded on 24-well glass slides. Then, broke the membrane with 0.3% PBST for 10 min and closed sections with 3% BSA for 2 h. Next, incubated the sections separately with anti-alpha-synuclein (Cat#ab280377, 1:500, Abcam, United Kingdom), anti-phospho S129 alpha-synuclein (Cat#ab51253, 1:500, Abcam), anti-S100β (Cat#ab52642, 1:500, Abcam), anti-NF-κB (Cat#8242, 1:500, CST, Boston, USA), and anti-TLR2 (Cat#ab209216, 1:500, Abcam) at 4 °C overnight. The following day, washed the sections with PBS 3 times and then incubated with secondary antibodies (Alexa Fluor conjugates, Abcam) for 1.5 h at room temperature. At last, the Hoechst (Cat#4082, 1:1000, CST) was stained. Images were taken with the laser scanning confocal fluorescence microscope (CarlzeissLSM710, Oberkochen, Germany).
Mice brain sections (20 µm) were incubated with 3% H₂O₂ for 15 min in the dark, which inactivated endogenous peroxidase. Secondary antibodies (Cat#SA00001-1, Cat#SA00001-2, 1:1000, Proteintech, Chicago, USA) were kept at room temperature for 1.5 h. After staining with DAB, dehydrated under gradient alcohol and sealed with neutral gum. Positive cells were counted serologically by Microbrightfield Stereo-Investigator software.