Immunostaining of Iba1 and Siglec-E in Brain Sections

Immunostaining was performed as we previously reported [47 (link), 48 (link)]. In brief, fixed coronal brain section (25 µm) underwent 60-min permeabilization and blocking with PBS containing 0.2% Triton X-100, 10% goat serum, and 22.52 mg/mL glycine. The sections were then incubated in rabbit anti-Iba1 polyclonal antibody (Wako cat. #019-19741, 1:200) and rat anti-Siglec-E monoclonal antibody (BioLegend cat. #677102, 1:100) at 4 °C overnight. Slices were then rinsed and incubated with anti-rabbit and anti-rat secondary antibodies conjugated with Alexa Fluor 546 or 488 (1:1000, Invitrogen) for 2 h. The slices were carefully mounted onto slides using the ProLong™ Gold antifade reagent (Invitrogen). The digital images were captured using a fluorescence microscope BZ-X800 (Keyence). The image processing and quantitative analyses were performed using the ImageJ/Fiji software (NIH).