Rabbit anti aβ42

Brains were dissected and immediately fixed in 5 volumes of PLP fixative (4% paraformaldehyde, 75 mM lysine, 10 mM sodium periodate; pH 7.4) at 4°C overnight and cryoprotected using 10% and 20% glycerol/2% dimethylsulfoxide, in 0.1 M PBS, pH 7.3 (24 h each). Serial, frozen sections (40 µm, coronal) were cut with a sliding microtome from the anterior frontal pole of to the caudal occipital region. All sections intended to be subjects of comparative analyses were processed together and incubated for the same time periods in all of the reagents. For Aβ42 immunohistochemistry, sections were treated with>95% formic acid (Sigma) for 2 min with gentle agitation, washed with PBS, and then transferred to a solution of PBS/10% Goat Serum (Gibco) for 1 h at room temperature (RT). Sections were probed with rabbit anti-Aβ42 (1∶2500, Invitrogen) overnight at RT in 0.3% triton-X 100, 2% Goat Serum (Gibco), 0.008% sodium azide PBS. Following washing, sections were probed with goat anti-Rabbit-HRP (Millipore, 1∶1000) in a solution of 2% Goat Serum/PBS for 3 h at RT. After incubation in a developing solution containing diaminobenzidine (DAB), sodium imidazole, and hydrogen peroxide, sections were mounted on subbed slides. Photomicrographs were taken with 2X and 4X magnification objectives and images of the hippocampus were analyzed with ImageJ software (NIH).

The antibodies used for immunofluorescence experiments were rat anti-LAMP1 (#1D4B, DSHB), rabbit anti-Aβ42 (#700254, Invitrogen), rabbit anti-BACE1 (#ab108394, Abcam), rabbit anti-βIII tubulin (#ab18207, Abcam). For immunoblotting, the antibodies used were rabbit anti-actin (#926–42,210, LI-COR), rabbit anti-p44/42 MAPK (pErk1/2) (#CS9101, Cell Signaling Technology), rabbit anti-44/42 (Erk1/2) (#CS137F5, Cell Signaling Technology), rabbit anti-LAMP1 (#CS3234, Cell Signaling Technology), mouse anti-BACE (3D5; Zhao et al., 2007), mouse anti-APP (6E10) (#803001, BioLegend), rabbit anti-p-tau (ser404) (#CS20194, Cell Signaling Technology), mouse anti-Tau1 (#MAB3420, Millipore Sigma), prelamin-A, (#MABPT858, Millipore Sigma) and mouse anti-HDJ-2 (#sc-59,554, Santa Cruz). Anti-rabbit or -mouse secondary antibodies from Vector Laboratories were used for immunoblots.