Alexa fluor 568 conjugated anti mouse secondary antibody

We washed coronal brain sections (40 μm) from experiment 1 in 1× PBS, blocked with 3% normal goat serum (NGS) in 1× PBS with 0.25% Triton X-100 (PBS-Tx), and incubated 24 h at 4°C with anti-Fos antibody (1:5000 dilution; Cell Signaling Technology catalog # 5348, RRID: AB_10557109) in blocking solution. We then washed sections in 1× PBS, and incubated them with Alexa fluor 488 conjugated goat anti-rabbit secondary antibody (1:500 dilution; Thermo Fisher Scientific catalog #A-11 008, RRID: AB_143165) and Alexa Fluor 568 conjugated anti-mouse secondary antibody (1:500 dilution; Thermo Fisher Scientific catalog #A-1104, RRID: AB_141371). Fluorescent images of immunoreactive cells in the IL, PL, and NAc were captured using a Keyence BZ-X810 microscope at 20× magnification with BZ-X Analyzer software. Cell profiler was used to automatically count total number of cells labeled with NeuN from two sections (bilateral) per rat (three images per rat). Number of Fos-positive nuclei in these images were manually counted by observers blind to the test conditions (interrater reliability: Pearson’s correlate r = 0.93). We averaged the counts so that each rat was an n of 1 for each brain area. The percentage of total average Fos counts out of total average NeuN counts for each rat was the dependent measure for Fos expression for each region.

Sections were thoroughly rinsed with 0.1 M PBS, pH 7.4 and incubated in 1% glycine solution for 10 min and blocked in 10% normal goat serum in PBS with 0.1% Triton X‐100 for 30 min at RT. To detect microglia, astrocytes, and hAβ, subsequent sections were incubated overnight at 4°C with rabbit anti‐ionized calcium binding adaptor molecule 1 (Iba‐1) (1:1000, Wako Chemicals), mouse anti‐glial fibrillary acidic protein (GFAP) antibody (1:1000, Millipore), and mouse anti‐hAPP/hAβ antibody (1:3000; 6E10, Biolegend), respectively. After rinsing with PBS, sections were incubated with Alexa Fluor 568‐conjugated anti‐mouse secondary antibody (1:500, ThermoFisher Scientific) for 1 h at RT and then rinsed again with PBS. The sections were mounted and coverslipped with a fluorescent mounting medium (Dako).

Immunofluorescence was performed according to the manufacturer's instructions [40 (link)]. Cells grown in a 4-well chamber slide (Nalge Nunc, Rochester, NY) were washed two times with PBS and fixed with 4% paraformaldehyde for 30 min at RT. They were permeabilized with 0.5% Triton X-100 in PBS for 5 min at RT. The cells were covered with PBS containing 3% bovine serum albumin for 1 h at RT to block nonspecific hybridization and were then incubated with rabbit anti-vimentin, anti-N-cadherin, anti-phospho-GSK3β (Ser9), anti-β-catenin or anti-phospho-LRP6 at a 1:500 dilution ratio. After being washed with PBS, the cells were stained with an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (A11008, Life Technologies, Carlsbad, CA), an Alexa Fluor 568-conjugated anti-rabbit secondary antibody (A11011, Life Technologies) or an Alexa Fluor 568-conjugated anti-mouse secondary antibody (A11004, Life Technologies) at a 1:100 dilution ratio. Nuclei were counterstained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) (H-1200, Vector Laboratories, Burlingame, CA).