3,3-Diaminobenzidine immunohistochemistry

To detect CD11b-positive cells in brain tissue, 3,3-diaminobenzidine (DAB) immunohistochemistry was performed according to a previously described protocol [54]. The following antibodies were used: mouse α-CD11b (OX42) (1:3000; Bio-Rad; Hercules, CA) and horse α-mouse (1:300; Vector-Labs; Berlingame, CA). Slides were cover slipped with DPX medium (BDH Laboratories, Poole, England) and images were acquired with a Nikon Eclipse Ti microscope interfaced with NIS Elements Imaging Software (Nikon).

DAB staining of spleen tissue was also performed according to a previously described protocol, albeit with minor modifications. Briefly, cryopreserved spleen tissue sections (30 μm) were dried at 37 °C, rehydrated with PBS (pH 7.4), and permeabilized for 1 h containing 10% goat serum, 0.3% 1 M Lysine, and 0.3% Triton-X-100. Following permeabilization, spleen sections were treated for 40 min with 3% H₂O₂ to quench endogenous peroxidase activity. Sections were incubated overnight in the following antibodies: rabbit α-LIFR (1:200; Santa Cruz; RRID:AB_2136015) and mouse α-FoxP3 (1:5000; Abcam; RRID:AB_447114). Secondary detection was achieved using goat α-rabbit and goat α-mouse biotinylated secondary antibodies (1:300; Vector Labs). Slides were mounted with DPX medium (BDH Laboratories) after dehydration with ethanol and clearing with xylenes. All images were acquired using a Nikon Eclipse Ti microscope interfaced with NIS Elements Imaging Software (Nikon).