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 LAMP115 (link),69 (link)–71 (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).
Sections were immunolabeled for amyloid-beta (Aβ), microglia (Iba1), astrocytes (GFAP), tyrosine hydroxylase (TH), and a widely used marker for neuritic damage LAMP115 (link),69 (link)–71 (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).
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