Abc elite solution

Free-floating brain sections were washed in phosphate-buffered saline (PBS; Gibco Invitrogen, Waltham, MA, USA) for 20 min. Sections were then incubated in target retrieval solution (Dako, Glostrup, Denmark) at 85°C for 40 min. Afterward, sections were washed for 20 min in PBS, followed by blocking of endogenous peroxidases (3% H₂O₂ in PBS, Sigma Aldrich) for 10 min and then washed with PBS containing 0.02% Triton-X-100 (PBS-T; Sigma Aldrich). Next, sections were incubated with polyclonal primary rabbit anti-GFAP (1:500; Dako), rabbit anti-Iba1 (1:1,000; Sigma), rabbit anti-CD31 (1:500, Abcam), or polyclonal rabbit anti-AQP4 (1:500; Boster Biological Technology, Ref#PB9475) overnight at 4°C. The next day, sections were washed in PBS-T and subsequently incubated in polyclonal secondary biotinylated goat-anti-rabbit antibody (1:250; Vector Laboratories, Olean, NY, USA) in PBS-T for 2 h at room temperature. Sections were washed in PBS-T followed by incubation in ABC elite solution (1.5% solution A + 1.5% solution B in PBS, Vector Laboratories) for 1 h at room temperature. Afterward, sections were washed in PBS-T for 20 min, and immunolabeling was performed by using 3,3-diaminobenzidine solution (Acros Organics, Geel, Belgium). Sections were washed in distilled H₂O and PBS; mounted on gelatin-coated slides; dehydrated in 95%, 99% alcohol, xylene; and coverslipped.

Mice were anesthetized with isoflurane (Wako) and each organ was isolated. Organs were embedded in OCT Compound (Sakura Finetek Japan, Tokyo, Japan) in liquid nitrogen. Tissue sections (7 μm) were obtained using a cryostat (CM1800, Leica Microsystems, Wetzlar, Germany) and mounted on poly‐L‐lysine (PLL)‐coated slides. Tissue sections were fixed by 4% PFA and treated with Block Ace overnight. Sections were incubated with 10 μg/mL 38M or 64R for 60 minutes at RT. After a 5‐minute treatment with 3% H₂O₂ in methanol, sections were incubated with 1:1000 diluted biotinylated rabbit anti‐rat IgG (H+L; Jackson ImmunoResearch) for 60 minutes at RT. They were subsequently reacted with 1:100 diluted Elite ABC solution (Vector) for 30 minutes and substrate 3,3′‐diaminobenzidine (DAB, Dojin, Chemicals, Kumamoto, Japan) solution. Finally, they were counterstained with Methyl Green (Merck, Darmstadt, Germany). The localization of antibody‐defined components (LYVE‐1) was observed using a Zeiss Axiolab microscope (Carl Zeiss, Oberkochen, Germany).