Age- and sex-matched PGRN-deficient mice and their WT littermates were anesthetized with sodium pentobarbital and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (PFA) in PBS, pH 7.4. The brains were dissected out, postfixed in 4% PFA for 24 h, and cryopreserved in 30% sucrose/PBS for 48 h. Snap-frozen brains were embedded in tissue embedding freezing medium and sectioned at a 40-µm thickness using a cryostat. Coronal sections were collected in PBS and processed free floating for immunohistochemistry.
Coronal brain sections spanning the cortex, thalamus, and hippocampus were washed in PBS, and endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide/10% methanol solution for 10 min. Sections were washed in PBS and blocked in 10% normal goat serum (NGS), 0.1% Triton X-100 in PBS for 1 h at room temperature. Next, sections were incubated with various antibodies: rabbit anti-PGRN (1:1,000; Zhu et al., 2002 (link)), rat monoclonal anti-CD68 (1:500; AbD Serotec), mouse monoclonal antiubiquitin (1:50,000; Millipore), rabbit polyclonal anti-GFAP (1:2,000; Dako), mouse monoclonal anti–phospho–TDP-43 (pS409/410; 1:500; provided by H. Akiyama, M. Hasegawa, and T. Arai, Tokyo Institute of Psychiatry, Tokyo, Japan; Inukai et al., 2008 (link)), and rabbit polyclonal anti–TDP-43 (Proteintech) in 2% NGS, 0.01% Triton X-100 in PBS for 16 h at 4°C. Sections were washed in PBS and incubated with appropriate biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) in PBS for 1 h at room temperature. After incubation in streptavidin ABC enhancer solution (Vector Laboratories) for 30 min at room temperature, immunostaining was visualized with diaminobenzidine (DAB; Sigma-Aldrich) as the chromogen. Sections were washed and mounted on precoated slides (Superfrost Plus; VWR) and allowed to air dry. The sections were further counterstained with either cresyl violet acetate or thionin (Sigma-Aldrich). The slides were dehydrated in ascending series of ethanol, passed through xylene, and coverslipped with DPX mounting media (Electron Microscopy Science). Images were acquired using a digital camera (Coolpix 5000; Nikon).
Coronal brain sections spanning the cortex, thalamus, and hippocampus were washed in PBS, and endogenous peroxidase was quenched by incubation in 3% hydrogen peroxide/10% methanol solution for 10 min. Sections were washed in PBS and blocked in 10% normal goat serum (NGS), 0.1% Triton X-100 in PBS for 1 h at room temperature. Next, sections were incubated with various antibodies: rabbit anti-PGRN (1:1,000; Zhu et al., 2002 (link)), rat monoclonal anti-CD68 (1:500; AbD Serotec), mouse monoclonal antiubiquitin (1:50,000; Millipore), rabbit polyclonal anti-GFAP (1:2,000; Dako), mouse monoclonal anti–phospho–TDP-43 (pS409/410; 1:500; provided by H. Akiyama, M. Hasegawa, and T. Arai, Tokyo Institute of Psychiatry, Tokyo, Japan; Inukai et al., 2008 (link)), and rabbit polyclonal anti–TDP-43 (Proteintech) in 2% NGS, 0.01% Triton X-100 in PBS for 16 h at 4°C. Sections were washed in PBS and incubated with appropriate biotinylated secondary antibodies (Jackson ImmunoResearch Laboratories, Inc.) in PBS for 1 h at room temperature. After incubation in streptavidin ABC enhancer solution (Vector Laboratories) for 30 min at room temperature, immunostaining was visualized with diaminobenzidine (DAB; Sigma-Aldrich) as the chromogen. Sections were washed and mounted on precoated slides (Superfrost Plus; VWR) and allowed to air dry. The sections were further counterstained with either cresyl violet acetate or thionin (Sigma-Aldrich). The slides were dehydrated in ascending series of ethanol, passed through xylene, and coverslipped with DPX mounting media (Electron Microscopy Science). Images were acquired using a digital camera (Coolpix 5000; Nikon).
