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 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.
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