Anti s100a10

Paraffin-embedded colon sections were deparaffinized in xylene and rehydrated through graded alcohol to water. After unmasking antigens by 0.01 mol/L citrate buffer solution, the colon sections were blocked with 2% BSA and stained with anti-Myelin basic protein (MBP) (Santa Cruz, USA), anti-Neuronal Nuclei (NeuN)(Abcam), and anti-GFAP (Abcam), anti-C3 (Abcam), and anti-S100A10(Abcam) primary antibodies overnight at 4°C. Signals were determined using FITC-conjugated secondary antibodies (Boster Biological Technology) and then counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (Abcam). The catalog numbers and dilutions of each antibody are shown in Table 1. Images were collected on a Leica TCS SPS microscope (Wetzlar, Germany), for fluorescence microscopy was used for nuclear counterstaining. ImageJ software was used to analyze fluorescence intensity in colon sections statistically.

After having been cut to yield 4 μm sections, tissue sections were deparaffinized with xylene (Beijing Chemical Plant, China), rehydrated with an ethanol gradient (Beijing Chemical Plant), and treated with 3% H₂O₂ (Beyotime, China) prior to antigen retrieval at room temperature (RT). Sections were blocked for 30 min at RT using 5% BSA (Abcam, UK), incubated overnight with anti-S100A10 (Abcam) at 4°C, probed for 30 min using a secondary antibody (Abcam), and color was developed using DAB (Abcam) prior to hematoxylin counterstaining (Abcam), ethanol gradient dehydration, neutral resin fixation, and imaging via light microscopy.

Western blot analysis was performed as previously reported (Jiang et al., 2016 (link); Bier et al., 2018 (link)). Briefly, cell lysates were solubilized with RIPA buffer supplemented with PhosSTOP and Complete Phosphatase/Protease Inhibitor Cocktails (Roche Diagnostics) and protein content was analyzed using a standard BCA assay. Protein extract (20–30 μg per sample) were loaded on sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred to PVDF membranes that were probed with the specific antibodies as detailed. Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Pharma-Biotech). Equal loading was verified using an anti actin antibody. The following primary antibodies were used: Anti-TrkB (Cat# sc-136990, 1:500), anti-p75 (Cat# sc-13577, 1:1,000), and anti-EAAT2 antibodies (Cat# sc-365634, 1:500) (Santa Cruz Biotechnology). Anti-cleaved PARP1 (AB3820, 1:1,000), Anti-cleaved caspase 3 (ab2303, 1:500), anti-C3 (ab97462, 1:500) anti-S100A10 (ab76472, 1:500) and exosome panel antibody (ab275018) (Abcam, Cambridge, MA United States). Anti-mouse and anti-rabbit HRP secondary antibodies (1:10,000, Pierce).