As026

Rice nuclei isolated from formaldehyde-fixed rice leaves were spread on microscope slides for specimen preparation. Rabbit anti-H3K4me3 (ABclonal, A2357) and mouse anti-H3K9me2 antibodies (Abcam, ab1220), which were used at a 1:200 dilution, were detected with donkey anti-rabbit IgG-Alexa 488 (1:100 dilution, ABclonal, AS035) and goat anti-mouse IgG-TRITC antibodies (1:100 dilution, ABclonal, AS026), respectively. The specimens were counterstained with vectashield antifade mounting medium (Vector, H-1220) and observed under a Nikon N-SIM microscope with 3D SIM mode to scan all the immunofluorescence at different z-axis in the whole nuclei. Spot function of Imaris X64 software (www.bitplane.com) was applied to count the foci with an estimated diameter of 0.15 μm and a threshold on “quality”, allowing an accurate detection of visible foci without including the background signal. A total of 22 nuclei were analyzed and averaged for foci counts.

We used the amount of microglia-phagocytosing synaptosomes and TRITC-Dextran 40 kDa (AS026, Abclonal) (Xue et al., 2022 (link)) to assess the changes in the phagocytosis of microglia post CX3CL1 and hypoxia treatment. The synaptosome mixture was centrifuged at 150,000 × g for 30 min and the precipitate was resuspended in cell medium. Thereafter, primary microglia were separately incubated with 100 μg/mL TRITC-Dextran 40 kDa or synaptosome mixture for 30 min. The cells were then fixed and stained with a synaptosomal marker Synaptophysin antibody (101011, Synaptic Systems) using immunofluorescence. Images were recorded by a Leica SP8 confocal microscope to quantify the fluorescence intensity of synaptosomes or TRITC-Dextran in the cells.