Vector sg blue

After 2 days fixation in 4% paraformaldehyde (PFA), the half brains were incubated for 48 h in 30% sucrose. Brains were then frozen in 2-propanol (Merck) and subsequently sectioned on a freezing-sliding microtome to collect 25 μm coronal sections. For histological staining, brain sections were stained with Thioflavin T dye according to standard protocol (ThT 1% in 50% Ethanol; Sigma Aldrich, St. Louis, MO). Sections were immunostained to visualize Aβ deposits using mouse monoclonal antibody (6E10, 1:500; COVANCE). Microglial reaction was assessed using a rabbit polyclonal antibody to Iba-1 (1:1000; Wako, 019–19141). Vectastain Elite ABC Kits were used and revelation obtained with Vector SG Blue (Vector Laboratories).

Frozen sections (20 μm) were first incubated with goat anti-mouse serpinA3N (R&D Systems cat. no. AF4709) at a dilution of 1:1000 overnight at 4 °C then incubated for 30 min at room temperature with biotinylated anti-goat IgG (Vector Labs cat. no. PK-6105) according to the manufacturer’s instructions followed by ABC reagent (Vector Labs cat. no. PK-6105) and stained with 3,3′-diaminobenzidine (DAB) substrate solution (brown) (Vector Labs cat. no. SK-4100). Sections were then thoroughly washed and incubated with either chicken anti-GFAP (Millipore, UK cat. no. AB5541), rabbit anti-IbA1 (Wako cat. no. 019-19741), rabbit anti-NPY (ThermoFisher Scientific cat. no. PA5-19568) or rabbit anti-AgRP (Phoenix Pharmaceuticals cat. no. H-003-57) at a dilution of 1:300 overnight at 4 °C and then at room temperature for 30 min with either goat anti-chicken IgG (Vector Labs cat. no. BA-9010) at a dilution of 1:200 followed by Vectastain Elite ABC kit (goat IgG) (Vector Labs cat. no. PK-6105) or directly with the Vectastain Elite ABC kit (goat IgG) and stained with Vector SG (blue) (Vector Labs cat. no. SK-4700). Control sections omitted the primary antibodies.