Sig 39320

At 8 DIV, cells were washed with ice-cold PBS and fixed in a solution of 4% paraformaldehyde for 20 min at room temperature (RT) and washed twice with PBS. Cells were then treated with 0.1 M PBS, 0.3% Triton X-100 containing 1% BSA and 1% normal blocking serum prepared from the species in which the secondary antibody was raised for 1 h at RT.
The following primary anti-sera were used: anti-6E10 (mouse; Covance, SIG-39320, batch no. D11AF00145; 1:1000), anti-β-III-tubulin (mouse; R&D Systems, MAB-1195, batch. no. HGQ0113121; 1:1000), anti-GFAP (rabbit; Dako, Z0334, batch no. 20005461; 1:1000) overnight at 4°C. Cells were washed with PBS and incubated with goat Alexa 488-conjugated anti-mouse, goat Alexa 568-conjugated anti-rabbit, goat Alexa 658-conjugated anti-mouse (Invitrogen) secondary antibodies for 30 min at 37°C. Cells were washed twice in PBS and incubated with the nuclear dye Hoechst 33258 (1 µg/ml) for 20 min at RT, washed with PBS and mounted with 0.1% para-phenylendiamine solution.

Tissue sections were deparaffinized in xylene and rehydrated in a gradient series of ethanol. The sections were heated in a citrate buffer (pH 6.0) in boiling water for 30 min for antigen retrieval. For blocking nonspecific staining, the slides were incubated with 1.5% normal horse serum (Vector Laboratories Inc., Burlingame, CA, USA) for 30 min, and then incubated with the following primary antibodies overnight at 4 °C: mouse anti-β-amyloid (6E10, 1:2000, sig-39320, Covance, Princeton, NJ, USA), anti-GFAP (1:1000, z0334, Dako, Carpinteria, CA, USA), and anti- Iba1 (1:1000, 019-19741, Wako, Osaka, Japan) antibodies. Subsequently, the slides were washed with 0.1% TritonX-100 PBS-T and incubated with biotinylated secondary antibodies (Vector Laboratories Inc.) for 30 min at room temperature. Slides were then incubated with an avidin–biotin–peroxidase complex (Vector Laboratories Inc.) for 30 min at room temperature. After incubation with diaminobenzidine (DAB, Vector Laboratories Inc.), images were obtained at 20× magnification using whole slide digital scanning with a digital pathology scanner (Aperio VERSA; Leica Biosystems, Richmond, IL, USA).

The hippocampi were homogenized in a pro-prep solution (17081, iNtRon, Gyeonggi-do, Korea) after physically mincing. The protein concentration was quantified using the Bradford reagent (Bio-Rad, Hercules, CA, USA), and then denatured by boiling for 5 min, depending on the concentration of the sample. Each sample was separated by electrophoresis on 10–15% sodium dodecyl sulfate-polyacrylamide gels. Then, the proteins were transferred to a nitrocellulose membrane and blocked for 30 min with 5% bovine serum albumin (BSA) solution for nonspecific blocking. Membranes were incubated at 4 °C with anti-6E10 (1:1000, Sig-39320, Covance), anti-ADAM10 (1:1000, ab1997, Abcam, Cambridge, UK), anti-BACE (1:1000, sc-33711, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFAP (1:1000, ab53554, Abcam), anti-Iba1 (1:1000, ab15690, Abcam), or anti-β-actin (1:3000, #3700, Cell Signaling, Danvers, MA, USA). After overnight incubation, membranes were washed thoroughly in PBS-T buffer and incubated for 1 h with the corresponding secondary antibody diluted at 1:3000. The membranes were then washed, and immunoreactivity was detected using an enhanced chemiluminescence (ECL) kit (NEL105001EA, Perkin Elmer, Waltham, MA, USA).

After behavioural tests, the brains of the wild‐type and APP/PS1 mice were removed and fixed in 4% paraformaldehyde (pH 7.4). The fixed brain samples were then immersed in 30% sucrose for cryoprotection and cut into 30‐μm‐thick slices using a Cryostat (Microm HM 525, Thermo Scientific, Waltham, MA, USA). The sliced brains were stained with 500 μM of thioflavin S (ThS) dissolved in 50% ethanol for 7 min. The sections were then rinsed with 100, 95, 70% ethanol and PBS successively. For immunostaining, the slides were stained with anti‐Aβ monoclonal antibody (1:100 dilution, clone 6E10, Catalog SIG‐39320, Covance), anti‐GFAP polyclonal antibody (1:300 dilution, Catalog AB5541, Millipore Corporation) and anti‐tau (phospho‐Ser199) antibody (1:100 dilution, Catalog ab81268, Abcam). Alexa Fluor 488‐ or Alexa Fluor 568‐conjugated secondary antibodies (Life Technologies) were used for fluorescence detection. Hoechst 33342 (Sigma‐Aldrich) was used for the visualization of nuclei. Images were taken on a Leica DM2500 fluorescence microscope (Li et al, 2007).

Immunofluorescent staining of oligomeric Aβ by C6T and A4 was performed as follows. In brief, the human or mouse brain sections were incubated with purified scFv antibody A4 and C6T (1 μg/ml) at 4 °C overnight. Then, a rabbit anti-cMyc antibody (C3956; 1:1000 dilution; Sigma–Aldrich) was used to detect the cMyc tag on the C6T and A4 scFvs. Images were double stained with antibody 6E10 to label Aβ (SIG-39320; 1:2000 dilution; Covance) or an antibody against MAP2 as a neuronal marker (MMS-485P; 1:500 dilution; Covance). Sudan black was used for quenching nonspecific fluorescence of intracellular lipofuscin. A control using secondary antibody against Myc tag without the corresponding primary scFv was also performed (Fig. 1). Microglial cells were visualized with rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1) antibody (catalog no.: 019-19741; 1:500 dilution; Wako). The synapses were immunostained using a rabbit antibody against SYP (Santa Cruz; 1:100 dilution), and newborn neurons in the hippocampus were labeled with rabbit anti-DCX (Ab18723; 1:500 dilution; Abcam). Fluorescent-conjugated secondary antibodies were used to visualize the target structures. The endogenous lipofuscin was blocked with 0.3% Sudan black, and cell nuclei were labeled with 4',6-diamidino-2-phenylindole (Electron Microscopy Sciences).