Igg free albumin

The half of each brain obtained as described above was fixed in 4 % buffered formalin at 4 °C overnight. Free-floating 30 μm-thick mouse brain sections were processed as previously described [33 (link)]. After antigen retrieval by incubating in citrate buffer (0.01 M citric acid, 0.05 % Tween 20, pH 6.0) at 70 °C for 50 min, samples were thoroughly washed several times with TBS and blocked with a solution of 2 % IgG-free albumin (Sigma) in TBS for 20 min at room temperature. Brain sections were then incubated overnight at 4 °C with rabbit anti-GFAP (Invitrogen) or anti-Iba-1 (WAKO, VA) polyclonal antibody in TBS-2 % BSA to detect astrocytes or microglia, respectively. After washing, sections were incubated for 1 h at room temperature with AlexaFluor 594 goat anti-rabbit IgG (Molecular Probes, OR) diluted in TBS-2 % BSA. Samples were mounted onto glass slides in Vectashield medium (Vector Laboratories, CA) containing DAPI for nuclei imaging. Samples were viewed on an Olympus Ix51 microscope equipped with a DP71 camera (Nikon Instruments Inc., NY).

Blocks of temporal cortex from AD (n = 6) and control (n = 6) brains were fixed by immersion in a solution of 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4, at 4 °C. Sections of 50 μm in thickness were cut on a sliding microtome (Jung Histoslide 2000R; Leica, Heidelberg, Germany). Antibody epitopes were retrieved, following treatment in citrate buffer (0.1 M citric acid, 0.1 M sodium citrate, pH 6.0) at 100 °C, for 15 min. Tissues designated to assess Aβ were incubated with 70% formic acid, for 20 min, at room temperature. Non-specific sites were blocked with 0.2% IgG-free albumin (Sigma-Aldrich, St. Louis, MO, USA) in PBS, for 1 h, at room temperature. Tissues were then incubated with the primary antibodies cocktail (Table 1), overnight, at 4 °C, and then with the secondary antibodies for 1 h (Table 2). Sections were counterstained with thiazine red (TR), to identify beta-pleated sheet conformation [12 (link)], and with TO-PRO^®-3 or DAPI for staining nuclei. Slices were mounted in Vectashield antifade mounting medium (Vector Laboratories, Burlingame, CA, USA).