Ab190684

Neurons from NSCs were fixed for 20 min in phosphate-buffered saline (PBS) containing 4% formaldehyde, permeabilized with 0.05% Triton (5–10 min, 20°C), and processed for double-labeling with the appropriate antibodies. Secondary antibodies coupled to Alexa Fluor dyes (488 or 594) were obtained from Invitrogen (Hellerup, DK). The nuclei were visualized by staining with DAPI (1 μg/mL; Sigma-Aldrich, Hinnerup, DK). Digital images were obtained with a Zeiss LSM lsm780 confocal system using 63 × oil NA 1.3 objectives (Carl Zeiss, Oberkochen, Germany). The colocalization results were quantified using the Zen software, following the procedures previously reported by La Rosa et al. [29 (link)], and Pearson's coefficient (R coefficient) was used as colocalization coefficient.
For immunofluorescence analysis, we used mouse anti-Aβ (ab11132) and rabbit anti-APP (clone Y188, ab32136), mouse anti-EEA1 (ab70521), mouse anti-Giantin (ab37266), mouse anti-SORL1 (ab63336), rabbit anti-SORL1 (ab190684) and rabbit anti-MAP-2 (ab32454), rabbit anti-β III Tubulin antibody (ab18207) and anti-GAP43 (ab16053), anti-GFAP (ab7260) and NeuN (clone 1B7) from Abcam (Cambridge, UK).

Dissected hippocampal tissues or cell samples were added to lysis buffer (Solarbio, Beijing, China), then ultrasonicated and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and protein quantification was conducted via the Lowery method. Fifty micrograms of total proteins were added to a 10% SDS-PAGE gel (Solarbio, Beijing, China), separated by electrophoresis, and transferred to polyvinylidene fluoride membrane, which was blocked with 5% skim milk (Solarbio, Beijing, China). Membranes were incubated with primary antibodies SORLA (ab190684; Abcam, Cambridge, UK), Aβ (ab2539; Abcam, Cambridge, UK), Phospho-Tau (#20194; Cell Signaling Technology, USA), and GAPDH (#5174; Cell Signaling Technology, USA) were incubated overnight at 4° C. Secondary antibody (#7074; Cell Signaling Technology, USA) were added, and the ECL method was used to measure luminescence. X-ray scanning was used for imaging, and Seion-Image software was used for quantitative analysis of the results.