Ab150160

The following main reagents were used: Aβ25–35 fragment (Sigma, United States), CEF (Roche, Switzerland), a mouse monoclonal antibody to Aβ (cat. # NBP2-13075, 1:1,000 dilution; Novus Biologicals, United States), a rat monoclonal antibody to CD54/ICAM-1 (cat. # 16-0542-81, 1:300 dilution; eBioscience, Thermo Fisher Scientific, United States), a goat polyclonal antibody to microglial marker AIF-1/IBA1 (cat. # NB100-1028, 1:200 dilution; Novus Biologicals, United States), an Alexa Fluor 568-conjugated goat anti-mouse IgG polyclonal antibody (cat. # ab175473, 1:400 dilution, Abcam, United Kingdom), an Alexa Fluor 594-conjugated goat anti-rat IgG polyclonal antibody (cat. # ab150160, 1:500 dilution; Abcam, United Kingdom), and an Alexa Fluor 488-conjugated donkey anti-goat IgG polyclonal antibody (cat. # ab150129, 1:200 dilution; Abcam, United Kingdom).

Fixed lungs were paraffin embedded, sectioned at 4 microns and mounted onto slides. Slides were subjected to H&E or Carstairs staining. For immunohistochemistry, slides were deparaffinized and rehydrated, followed by 20-min heat-induced antigen retrieval with Tris/EDTA pH 9. The slides were washed with TBS/0.025% Triton X-100 then blocked with 10% donkey or goat serum with 1% BSA in TBS. Slides were probed with the following primary antibodies: anti-CitH3 (ab5103; Abcam), anti-Ly6G (127602; Biolegend) and anti-MPO (ab9535; Abcam). The secondary antibodies used were donkey anti-rabbit IgG, Alexa 647 (ab150075, Abcam), AffiniPure donkey anti-rabbit IgG, Alexa 488 (711-545-152; Jackson ImmunoResearch Laboratories) or goat anti-rat IgG, Alexa 594 (ab150160; Abcam). Glass coverslips were mounted onto the slides using Vectashield antifade mounting medium with DAPI (H-1200; Vector Laboratories) and imaged with fluorescence or confocal microscopy.

Mouse lung tissues were fixed in 4% paraformaldehyde and serial 3-μm-thick paraffin-embedded sections were used for immunofluorescence. The samples were deparaffinized and rehydrated, and lung sections were blocked using 1% sheep serum at room temperature for 1 h. Next, the sections were incubated with anti-JMJD3 (1:200, NBP1-06640, Novus, USA), anti-H3K27me3 (1:200, ab192985, Abcam, Cambridge, UK), anti-C/EBPβ (1:200, 23431-1-AP, Proteintech, USA), anti-cleaved caspase 3 (1:200, 9664S, CST, Boston, USA), anti-F4/80(ab16911, Abcam, Cambridge, UK), anti-ADORA2A (1:200, ab34611, Abcam, Cambridge, UK), and anti-H3K27me3 (ab6002, Abcam, Cambridge, UK) antibodies overnight at 4 °C. The next day, the sections were incubated with the appropriate secondary antibodies (1:200, ab15007, ab150079, ab150160, and ab150115; Abcam, Cambridge, UK) for 2.5 h at room temperature. Subsequently, the samples were incubated with 4′,6-diamidino-2-phenylindole (DAPI) to label the nuclei. Besides, the cells in lung tissue with F 4/80+ were recognized as macrophages. Immunofluorescence was observed under a fluorescence microscope (Olympus, Tokyo, Japan). Different groups of lung tissue were observed in the same settings and differences among groups were compared. Image-Pro Plus was used for image merging and was semi-quantitatively analysed by the ImageJ software (NIH, Bethesda, MD, USA).