Anti 6e10

Coronal sections were rinsed with distilled water and stained with alum hematoxylin, followed by differentiation with 0.3% acid alcohol. After rinsing with distilled water, the sections were stained with eosin for 2 min and dehydrated for mounting. TUNEL assays (ThermoFisher Scientific) and Nissl staining (Servicebio, China) were performed according to the manufacturer’s instructions.
Immunostaining was performed using rabbit monoclonal anti-PARP16 (ARP33751, Aviva Systems Biology, USA) and anti-6E10 (800304, BioLegend). Normal rabbit IgG (2729, Cell Signaling Technology) was used as negative control. Secondary antibodies conjugated with horseradish peroxidase (ab64264, Abcam) were used and visualized according to standard protocols.

Brain samples were homogenized in ice‐cold RIPA lysis buffer. Protein samples were loaded on a 4%–20% SDS‐polyacrylamide gel and then transferred onto nitrocellulose (NC) membranes. After blocking with 5% fat‐free milk, the NC membranes were incubated with the following primary antibodies overnight at 4°C: anti‐APP C‐terminal (1:1000, Biolegend); anti‐6E10 (1:400, Biolegend); anti‐RAGE (1:1000, Millipore); anti‐LRP‐1 (1:1000, Abcam); anti‐pS396‐tau (1:1000, Abcam); anti‐pT231‐tau (1:1000, Signalway); anti‐Tau5 (1:1000, Millipore); anti‐Synapsin‐1 (1:1000, Abcam); anti‐PSD95 (1:1000, Millipore); and anti‐β‐actin (1:2000, Origene). The membranes were incubated with the corresponding IRDye 800 CW‐conjugated secondary antibodies and scanned using an Odyssey fluorescent scanner. Relative band intensities were normalized to the band intensity of the internal reference protein for analysis.

The tissues were homogenized in radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor and phosphatase inhibitor. The protein concentration was determined using Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of the total protein (10–30 μg) were electrophoresed on 8–12.5% sodium dodecyl sulfate (SDS)–polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated in 3% bovine serum albumin (BSA) solution for nonspecific blocking, followed by overnight incubation at 4 °C with the primary antibody. The following primary antibodies and dilutions were used: Anti-6E10 (cat no. SIG-39320; 1:1000 dilution; Biolegend), anti-Iba-1 (cat no. 106-20001; 1:1000 dilution; Wako), anti-synaptophysin (cat no. MAB-5258; 1:1000 dilution; Merck), anti-PSD-95 (cat no. GTX133091; 1:1000 dilution; GeneTex, Irvine, CA, USA), and anti-β-actin (cat no. A1978; 1:2000 dilution; Sigma-Aldrich) antibodies. Subsequently, the membranes were washed in PBS containing 0.1% Tween 20 and incubated with a 1:3000 dilution of horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. The membranes were then washed and developed using an enhanced chemiluminescence (ECL) kit (Perkin Elmer, Waltham, MA, USA).

For immunohistochemistry, sections (40μm thick) were pretreated with 3% H2O2/3% methanol in Tris-buffered saline (TBS) for 30 min, followed by a TBS wash. Sections were then incubated in TBS with 0.1% Triton X-100 (TBST) for 15 min, followed by TBST with 2% BSA (Sigma-Aldrich) for 30 min. Sections were incubated with anti-6E10 (1:1000; Biolegend, San Diego, CA, USA) in TBS + 5% normal horse serum overnight at 40 C. Sections were then incubated with the appropriate secondary biotinylated antibody (1:500) in TBS containing 2% BSA plus 5% normal serum for 1 hour at room temperature, followed by Vector ABC Kit and DAB reagents (Vector Laboratories, Burlingame, CA, USA) to visualize staining.

Mice were anesthetized by 3% isoflurane and perfused with phosphate-buffered saline (PBS, 0.01 M, pH = 7.4), then followed by 4% paraformaldehyde (PFA), and brains were post-fix overnight with 4% PFA. Brain tissues were sectioned into 30 μm-thick free-floating coronal or sagittal sections with different purposes using a vibratome (Leica VT1000S). All brain slices were sequentially collected and stored at -20 °C in cryoprotectant solution (FD Section Storage Solution) for further staining.
Free-floating sections were washed in PBS (3–5 min, 3 times) and incubated in blocking buffer (0.03% Triton X-100, and 2% donkey serum in PBS) for 30 min. Then the slices were incubated in primary antibody for overnight at 4 °C, washed 3 time in PBS in the next day, followed by incubating in secondary antibodies for 2 h at room temperature. Primary antibodies used were as follows: anti-VPS35, generated by Xiong lab as previously described [8 (link)]; anti-Iba1, ab178846, Abcam; anti-Iba1, ab5076, Abcam; anti-OC, AB2286, Sigma-Aldrich; anti-6E10, 803015, Biolegend; anti-ATG9A, ab108338, Abcam; anti-RTN3, 12055-2-AP, Thermofisher; anti-GFAP, 12389, Cell Signaling; anti-APOE, K74180B, Menidian Life Science; anti-TMEM119, ab209064, Abcam; anti-LPL, ab21356, Abcam; anti-Trem2, MAB2056, Abnova; anti-Clec7A, mabg-mdect, InvivoGen.

Free floating 30 µm sagittal brain sections were treated for antigen retrieval using sodium citrate buffer and subsequently stained using primary antibodies for Iba1 (1:250, rabbit, Wako) and anti-6E10 (1:1000, Biolegend), and the corresponding secondary antibodies and nuclear counterstain using DAPI. Images were acquired in the frontal cortex using a Zeiss confocal microscope with 40× magnification. Morphological analysis of microglial cells was performed using FIJI software. Each single microglial cell was identified in the Z-stack and a maximum intensity projection (MIP) was created for each individual cell from the image layers covering the individual microglial cell and the resulting image was binarized by thresholding. The area and the perimeter of the cell shape were measured and the CI was calculated (CI = 4π[area]/[perimeter]2) for each cell.
For analysis of ApoE expression in microglia and astrocytes, sections were stained with anti-P2Y12 (1:100, Anaspec) or anti-GFAP (1:200, DakoCytomation Z 0334), mouse anti-6E10 antibody (1:200, abcam), and biotinylated anti-ApoE antibody (1:50). Slides were then labeled with the secondary antibodies and counterstained with DAPI. Samples were imaged using an epifluorescence microscope (40× magnification) and co-localization of Αβ, ApoE, and microglia was analyzed using FIJI software.