Rabbit normal blocking serum

For immunofluorescence analysis, cells were transferred from U-bottom plates to gelatin-coated microscope slides by cytospine (300xg, 10 min) and perfused by PBS containing 4% (w/v) paraformaldehyde for 20 min at room temperature. Fixed cells were washed with PBS and blocked with 10% rabbit normal blocking serum (Santa Cruz Biotechnology, Dallas, TX, USA), supplemented with 0.3% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 45 minutes at RT. Next, cells were washed and double-stained for HSP70 (rabbit polyclonal antibody, 1:500, SPA-812, Enzo Life Sciences, Inc., Farmingdale, NY, USA) and AGO2 (mouse monoclonal antibody, 1:500, B-3, Santa Cruz Biotechnology, Dallas, TX, USA) with PBS supplemented with 1.5% of normal blocking rabbit serum and 0.3% Triton X-100, overnight at 4 °C in wet chamber. Subsequently, cells were washed and secondary fluorescent antibody (goat anti-rabbit IgG-TR and goat anti-mouse IgG-FITC; 1:100 both Abcam, Cambridge, UK), supplemented with 1.5% of normal blocking rabbit serum and 0.3% Triton X-100 was added for 1 h at RT. For fluorescent DNA nuclei staining, DAPI was used (1.5 mg/mL UltraCruz Mounteining Medium, Santa Cruz Biotechnology, Dallas, TX, USA). The intracellular colocalization of AGO2 and HSP70 was analyzed by confocal microscopy (Nikon D-Eclipse C1) using EZ-C1 software.

Astrocytes were transferred to gelatin-coated microscope slides by cytospin (300 × g, 10 min) and fixed with 4% paraformaldehyde for 20 min at RT. Fixed cells were washed extensively with PBS and blocked with 10% rabbit normal blocking serum (Santa Cruz Biotechnology) supplemented with 0.3% Triton™ X-100 (Sigma-Aldrich) for 45 min at RT. Next, cells were washed and double-stained for IgG1 with AQP4, C5b-9 with phalloidin, and GFAP with C5b-9. Anti-IgG1/FITC (Dako Cytomation), anti-AQP4 (Santa Cruz Biotechnology), anti-C5b-9 (Dako Cytomation), phalloidin (Invitrogen), anti-GFAP (Santa Cruz Biotechnology), and rat IgG2b (Invitrogen), as negative isotype control were used. Secondary fluorescent antibodies [chicken pAb to rabbit Texas Red (TR) (Santa Cruz Biotechnology), goat pAbs to mouse FITC (Abcam), or goat pAbs to mouse TR (Thermo Fisher Scientific)] were added. The membrane localization of C5b-9, IgG1, and GFAP was analyzed by confocal microscopy (Nikon D-Eclipse C1) using EZ-C1 software.