Ab289874

Staining was performed using standard immunohistochemical procedures as previously described.^[41 (link)
^] Sections and primary microglia were washed 3 times with 1 × PBS and blocked with 5% normal goat serum (Gibco) and 0.2% Triton X‐100 for 1 h. Then, sections were incubated with specific antibodies at 4 °C overnight: anti‐Sting (1:200; 19851‐1‐AP, ProteinTech), anti‐Mfn2 (1:200; ab124773, Abcam), anti‐Iba1 (1:200; ab289874, Abcam), and anti‐iNOS (1:200; ab178945, Abcam). 4′,6‐diamidino‐2‐phenylindole (DAPI) (1:1000, Invitrogen) was used to label the nuclei. All sections and microglia were imaged using a Nikon confocal microscope and analyzed using ImageJ software.

After a 72-hour post-SAH, mouse was sacrificed by an overdose of 2% pentobarbital sodium and underwent trans-cardiac perfusion with a 4% paraformaldehyde solution. Brain tissue was then removed and stored overnight in 4% paraformaldehyde. Dehydration was carried out using 30% sucrose solutions. Subsequently, brain sections measuring 15 μm were obtained and incubated in PBS solution containing 0.1% Triton X-100 for 30 min. To prevent non-specific binding, the sections were blocked with PBS solution containing 5% goat serum for an additional 30 min. Following this, the sections were incubated overnight at 4 °C with primary antibodies: IBA1 (ab289874, 1:300, Abcam) and CD68 (ab283654, 1:300, Abcam). Post-incubation, brain sections were thoroughly rinsed in PBS with three consecutive 5-minute washes. Subsequently, the sections were incubated at 25 °C for 1 h with corresponding secondary antibodies: Alexa Fluor 488 Anti-rabbit IgG (A11034, 1: 1000, Invitrogen) and Alexa Fluor 594 Anti-goat IgG (A11058, 1:1000, Invitrogen). Finally, the sections were stained with DAPI solution and observed using an A1 Si confocal microscope.