Rabbit anti β amyloid

Brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks. After deparaffinization and rehydration, brain slices were boiled in citric buffer (10 mM Citric acid, pH 6.0) for 4 min for antigen retrieval. The slides were incubated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Subsequently, sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti-β-Amyloid (1:400; Cell Signal Technology), mouse anti-Tau (1:1,000; Cell Signal Technology) and rabbit anti-P-Tau Thr181 (1:1,000; Cell Signal Technology). Following removal of the antibodies via several rinses with PBS, the respective antibodies were detected using the avidin-biotin-peroxidase complex method. Slides were lightly counterstained with hematoxylin and then were dehydrated with sequential ethanol. All images were analyzed by optical microscopy.

Equal amount of total protein samples were separated by SDS/PAGE (12% Bis-Tris gel; Life Technologies), and then transferred to PVDF membrane (BioRad Laboratories). After blocking in TBST buffer (20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween-20) containing 5% (wt/vol) nonfat dry milk (Santa Cruz) for 1 h at room temperature, the membrane was then incubated with primary antibodies overnight at 4°C. This was followed by incubation with the corresponding secondary antibody for 1h at room temperature. The following antibodies were used: mouse anti-Parkin antibody (1:500; Santa Cruz), rabbit anti-LC3B (1:1000; Cell Signaling Technology), rabbit anti- β-amyloid (1:6000; Cell signaling Technology), rabbit anti-TOM40 (1:1000; Santa Cruz), rabbit anti- MFN2 (1:6000; Cell signaling Technology), mouse anti-OPA1 (1:5000; BD Transduction Lab), mouse anti- DLP1 (1: 2000; BD Transduction Lab), and goat anti-mouse IgG HRP conjugated and goat anti-rabbit IgG HRP conjugated (1:6000; Life Technologies). Image J software (National Institutes of Health) was used to analyze the scanned blots and to quantify protein signal intensity.

The immunofluorescence staining of brain paraffin sections were performed as previously described [25 (link)]. After blocking with 5% bovine serum albumin, the sections were incubated with anti-MRTF-A (1:100, sc-398675, Santa Cruz, CA, USA), rabbit anti-β-amyloid (Cell Signaling Technology, 1:2000) or rabbit anti-LC3B (1: 500, ab48394, Abcam, MA, USA), respectively. Then, the sections were washed with 0.1M PBS and then incubated with the fluorescence-labeled secondary antibodies (goat anti-mouse/rabbit 1:200, ab150113, ab150077, Abcam, MA, USA) for 30 minutes at 37° C, and then washed with PBS. Next, the nuclei were stained with DAPI (5 μg/ml) for 2 minutes, and then analyzed with a laser scanning confocal microscope (CX31-32RFL, Olympus).