Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits - Nature Aging

. The fE/Cre mice were crossbred with tau-P301S transgenic mice PS19Vle/J) that express human P301S 1N4R tau driven by the PrP promoter to generate PS19-fE4 and PS19-fE3 mice with no Cre or Syn1-Cre. Littermates that were negative for Syn1-Cre were used as PS19-fE controls. For generation of the PS19-fE/Syn1-Cre line, only female Syn1-Cre mice were used for breeding purposes because germline recombination has been reported to occur in the progeny of male Syn1-Cre mice.

The PS19-fE mice were analyzed at 10 months of age. Brain tissue was collected after mice received intraperitoneal injections of avertin and were transcardially perfused with 0.9% saline for 1 min. Depending on the study, brain tissue was fixed as whole brains or hemi-brains. For hemi-brains, the right hemispheres were drop-fixed for 48 h in 4% paraformaldehyde , washed for 24 h in 1× PBS and cryoprotected in 30% sucrose for 48 h at 4 °C.

For DAB staining, several brain sections were washed in PBS-T and incubated for 5 min in boiling antigen retrieval buffer . Next, sections were washed in PBS-T and incubated for 15 min in endogenous peroxidase buffer and 3% H) and washed in PBS-T before being incubated in blocking solution for 1 h at room temperature. After blocking, sections were washed in PBS-T and incubated in avidin/biotin blockage for 15 min and then washed in PBS-T.

For Thio-S staining, several brain sections were mounted onto slides and the protocol was adapted from a previous study. The tissue was washed with 1× PBS-T and then incubated in a solution of 0.06% Thio-S in PBS for 8 min. Then, sections were washed for 1 min in 80% ethanol and 5 min in PBS-T. Sections were then counterstained with DAPI for 8 min, washed with PBS-T and coverslipped.

The hippocampus was dissected from snap frozen mouse hemi-brains after thawing on ice. The hippocampal tissue was weighed and homogenized using a Polytron immersion disperser homogenizer in ice-cold RAB buffer at 10 µl mgtissue, supplemented by phosphatase inhibitors and protease inhibitors . Samples were then centrifuged using an Optima TLX ultracentrifuge at 50,000for 20 min at 4 °C and the supernatant was collected as the RAB-soluble fraction.

Source: Healthcare Press (healthcarepress.net)

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