Ep38y

IHC was performed with a Ventana XT automated staining instrument. Antibodies recognizing the following targets were used: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Schweiz), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), HER2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell signaling, Danvers, MA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Sections were deparaffinized using EZ Prep solution (Ventana Corporation, Tucson, AZ). CC1 standard (pH 8.4 buffer containing Tris/borate/EDTA) was used for antigen retrieval and blocked with inhibitor D (3% H₂O₂) for 4 min at 37°C. Slides were incubated with primary antibody for 40 min at 37°C, followed by a universal secondary antibody for 20 min at 37°C. Slides were incubated in streptavidin-horseradish peroxidase (SA-HRP) D for 16 min at 37°C, after which the substrate, 3,3′-diaminobenzidine tetrahydrochloride (DAB) H₂O₂, was added for 8 min, followed by hematoxylin and bluing reagent counterstaining at 37°C.

Both WT and Npc1^−/− P7 microglia were cultured at a density of 5 × 10⁵ cells/well in a 12-well plate in microglia culturing medium. At 5DIV, microglia culturing medium was replaced with serum-free medium, containing only DMEM/F12 and 1% PenStrep. After 3 h of serum deprivation (t = 0), cells were treated with mouse recombinant EGF (40 ng/ml, R&D System) and cycloheximide (20 µg/ml, Sigma Aldrich) in serum-free medium. Cells were lysed at different time points (0, 1, 3, and 6 h) after treatment in STET lysis buffer and supplemented with protease and phosphatase inhibitors. EGF receptor (EGFR) level in each lysate was analyzed via western blot using an antibody against EGFR (anti-EGFR antibody EP38Y, Abcam). Each experimental group was tested in microglia isolated from three independent experiments (n = 3).

IHC was performed on a Ventana XT automated staining instrument (Ventana Medical Systems, Tucson, AZ, USA). The following target-specific antibodies were used according to the manufacturer's instructions and a previous study [20 (link)]: MutL homolog 1 (MLH1, ready to use, clone M1, Roche, Basel, Switzerland), MutS protein homolog 2 (MSH2, ready to use, clone G219-1129, Roche), MutS homolog 6 (MSH6, 1:100, clone 44, Cell Marque, Rocklin, CA, USA), postmeiotic segregation increased 2 (PMS2, 1:40, clone MRQ28, Cell Marque), ERBB2 (ready to use, clone 4B5, Roche), EGFR (1:100, EP38Y, Abcam, Cambridge, UK), c-MET (ready to use, clone SP44, Roche), PTEN (1:100, clone 138G6, Cell Signaling, Danvers, MA, USA), and p53 (1:300, DO7, Novocastra, Newcastle, UK). Epstein-Barr virus-encoded small RNAs (EBER) in situ hybridization (ISH) was performed using a Ventana Benchmark ISH system and ISH iView kit (Ventana Medical Systems, Tucson, AZ, USA).