Rat anti lamp1

293T, E0771, and JAWSII cells were grown on 8-well slides (MatTek) coated with 1 µg/mL human fibronectin (EMD Millipore). Transfection was performed using Lipofectamine3000 (Thermofisher). For Lysotracker (ThermoFisher) staining, cells were incubated with 75 nm Lysotracker for 1 hour at 37°C. For endoplasmic reticulum (ER) staining, cells were incubated with 2 μL ER CellLight ER-RFP Bacman V.2.0 (Thermofisher) for 24 hours. Slides were washed with phosphate buffered saline (PBS), fixed with 10% formalin for 20 min and mounted with DAPI Fluoromount-G (SouthernBiotech). Staining was performed in 0.1% PBST with rabbit anti-HER2 (1:200, Cell Signaling), rat anti-LAMP1 (1:200, Abcam) or rat anti-MHCII (1:200, Thermofisher) at 4° overnight and antirabbit Alexa Fluor 488 (1:1000 dilution, Invitrogen) or antirat Texas Red-X (1:1000, invitrogen) at room temperature for 1 hour. Slides were mounted with Fluoromount-G with DAPI (Southern Biotech).

Half-brains were fixed overnight in 4% PFA at 4°C, dehydrated in 30% sucrose overnight, and sectioned on a freezing stage microtome into 30 µm thick coronal slices stored in cryoprotectant solution. For immunofluorescence, sections were washed extensively in PBS and blocked with 10% bovine serum albumin (BSA, Sigma, A2153) for 1 h at room temperature (RT).
Sections were immunolabeled for amyloid-beta (Aβ), microglia (Iba1), astrocytes (GFAP), tyrosine hydroxylase (TH), and a widely used marker for neuritic damage LAMP1 ^{15 (link),34 (link)–36 (link)}; in different combinations specified in appropriate figure legends. The following primary antibodies were used: biotin anti-Aβ (clone 6E10, 1:3000, BioLegend), rabbit anti-Iba1 (1:2000, Wako), guinea pig anti-GFAP (1:3,000, Synaptic Systems), mouse anti-TH (1:500, Millipore Sigma) and rat anti-LAMP1 (1:2000, Abcam). Sections were incubated in primary antibodies for 48 h at 4°C. The sections were washed with PBS and incubated in fluorescently labeled secondary antibodies/reagents (Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 594, streptavidin conjugate 594 and Alexa Fluor 647, Invitrogen; all at 1:1000) for 4 hr at RT, then mounted and coverslipped (Prolong Diamond, ThermoFisher Scientific).