β3 tubulin

For immunofluorescence double staining, apoptosis was detected by the ApopTag Fluorescein In Situ Apoptosis Detection Kit (S7110; Chemicon International, Temecula, CA), as described previously^{15 (link)}. And then, sections were incubated overnight with cell surface glycoprotein F4/80 (Serotec, Oxford, UK) and a Texas red-labeled secondary antibody and counterstained with 4,6-diamidino-2-phenylindole (DAPI). We also performed immunofluorescence double staining for SOX10 (Abcam, Cambridge, UK), β3-tubulin (1:50; Cell Signaling Technology, Danvers, MA), and LC3 (1:200; Sigma-Aldrich, St. Louis, MO, USA). The fluorescent images were examined under a laser scanning confocal microscope system (Carl Zeiss LSM 700, Oberkochen, Germany).

Antibodies against JMJD2A, STMN1, β3-Tubulin, α-Tubulin and β-Tubulin were purchased from Cell Signaling Technology (Boston, USA). F-actin and PSA were purchased from Thermo Fisher (Shanghai, China), TUNEL Apoptosis Assay Kit from Beyotime (Shanghai, China), Phosphate-buffered saline (PBS) from Solarbio (Beijing, China), and Cell Counting Kit-8 from BBI Life Sciences (Shanghai, China).

The amyloid β protein fragment 1–40 (Aβ₁₋₄₀), GW4869, PKH67, LPS and IFN-γ were purchased from Sigma (California, USA). Lipofectamine 2000 were purchased from Invitrogen (CA, USA). The antibodies GAPDH, YB-1, PTEN, NeuN and Iba1 were purchased from Abcam (Cambridge, MA), and the antibodies β3 tubulin, CD206, CD11b, Arg-1, iNOS were purchased from Cell Signaling Technology (Boston, Massachusetts). The ELISA kits were purchased from Blue gene (Shanghai, China). The YB-1 plasmids, miR-223 antagomir and miR-223 agomir were purchased from Genechem (Shanghai, China). The RiboCluster Profiler RIP-Assay Kit was purchased from MBL Medical & Biological Laboratories Co (LTD, Japan).

For neuronal marker staining, sections were first repaired using ethylenediaminetetraacetic acid antigen retrieval solution (pH 9.0) for 5 min at 100°C and blocked with 3% BSA solution for 30 min. After rinsing, sections were probed overnight at 4°C with the following primary antibodies: β3-tubulin (1:200, Cell Signaling Technology) or Neurofilament-L (1:100, Cell Signaling Technology). Sections were then washed and incubated with Alexa Fluor 488 conjugated goat anti-rabbit or rabbit anti-mouse secondary antibodies (1:500, Invitrogen) for 1 h at 37°C, correspondingly.
To investigate the inflammatory response, immunofluorescence analysis of CD68 (1:1,000, Invitrogen) and CD206 (1:200, Proteintech) was performed similarly as earlier. Sections were then incubated with Alexa Fluor 647 conjugated goat anti-rabbit or Alexa Fluor 568 conjugated rabbit anti-mouse secondary antibodies (1:500, Invitrogen) for 1 h at 37°C, respectively.
To evaluate exosomes, CD63 primary antibody (1:200, Affinity) was selected and incubated with sections at 4°C overnight, then treated with Alexa Fluor 647 conjugated goat anti-rabbit secondary antibody.
Nuclei were counterstained with 4′,6-diamidino-2-phenylindole for 10 min. Sections were observed using a CLSM.