Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model methods
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Intracerebroventricular cannulation and pump implantation
Surgical implantation and cannulation were performed as described previously (Reichenbach et al, ). Briefly, mice were implanted s.c. at the interscapular region with osmotic minipumps (model Alzet 2006; delivery rate, 0.15 µl/h for 42 days; Durect) connected to Alzet Brain Infusion Kit 3 (anteroposterior [AP] -0.2 mm, medial lateral [ML] +1 mm relative to bregma; dorsal ventral [DV] +2.5 mm from the brain surface; Durect) for intracerebroventricular drug delivery. Animals were anesthetized with isoflurane (1.5% vol/vol) and O 2 (1 l/min) and kept on a heating pad (37°C) during surgery. Buprenorphine was used as an analgesic. Pumps were filled according to the manufacturer's instructions.
Immunohistochemistry
Sections stained for pStat3 were pre-treated for 45 min with 0.21% citric acid (90-95°C), followed by an incubation in 1% NaOH (w/v) and 2% H 2 O 2 (w/v) for 20 min, 0.3% glycine (w/v) for 10 min, and 0.03% SDS (w/v) for 10 min at room temperature. All brain sections were blocked with 10% normal goat serum (Vector Labs) and 0.3% Triton X-100 (Sigma) in PBS for 1 h. Mouse brain sections were incubated with rat anti-GFAP (1:250; #130300; Invitrogen), rabbit anti-GFAP (1:500; #Z0334, Dako), mouse anti-Aβ (IC16; 1:250; provided by Dr. C. Pietrzik, Mainz University), rabbit anti-pStat3 (1:500; #9145S, Cell Signaling), rabbit anti-Iba1 (1:250; 019-19741, Wako), rabbit anti-Stat3 (1:500; #12640, Cell Signaling), rabbit anti-RFP (1:300; #AB234, Evrogen), rat anti-LAMP1 (1:750; #121602, B...
Protein biochemistry
Mice were sacrificed, and one hemisphere was transferred to liquid nitrogen and stored at -80°C. Protein extraction was performed by homogenization in PBS (pH 7.4) with 1% phosphatase inhibitor and 1% protease inhibitor cocktails (Thermo Fisher) using a ceramic bead homogenizer (Precellys, VWR). Homogenates were extracted in RIPA buffer (in mM: 49.96 Tris, pH 7.2, 149.6 NaCl, 25.6 NP40, 24 NaDOC, 6.8 SDS) and centrifuged for 30 min at 100,000 g. The pellet containing insoluble Aβ was solubilized in SDS buffer (in mM: 69.2 SDS, 24.98 Tris, pH 7.5). Protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher) with a FLUOstar Omega Reader (BMG). Samples were solubilized in 4 × LDS buffer (Life Technologies), boiled for 10 min at 95°C, centrifuged for 5 min at 20,000 g at 4°C, and loaded on 4-12% NuPAGE No...
Data analysis
All immunohistochemical data points represent mean values of 5-10 brain sections per mouse. Confocal images were imported into ImageJ, converted to 8-bit images, and smoothed using a Gaussian filter. After contrast enhancement (0.4% saturated pixels), images were binarized using ImageJ (plaque stainings, automated MaxEntropy algorithm; GFAP and Iba1, a threshold defined as the mean background intensity plus the SD of background intensity multiplied by 2 was used). Astrocyte, microglia, and plaque area coverage as well as the number and size of plaques and LAMP1-positive dystrophic dendrites per area were quantified using ImageJ. The plaque area was subtracted from the area covered by dystrophic neurites before data analysis. Analysis of glial peri-plaque morphology was determined as described (Reichenbach et al, ). Briefly, following background removal, 3...
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Surgical implantation and cannulation were performed as described previously (Reichenbach et al, ). Briefly, mice were implanted s.c. at the interscapular region with osmotic minipumps (model Alzet 2006; delivery rate, 0.15 µl/h for 42 days; Durect) connected to Alzet Brain Infusion Kit 3 (anteroposterior [AP] -0.2 mm, medial lateral [ML] +1 mm relative to bregma; dorsal ventral [DV] +2.5 mm from the brain surface; Durect) for intracerebroventricular drug delivery. Animals were anesthetized with isoflurane (1.5% vol/vol) and O 2 (1 l/min) and kept on a heating pad (37°C) during surgery. Buprenorphine was used as an analgesic. Pumps were filled according to the manufacturer's instructions.
Sections stained for pStat3 were pre-treated for 45 min with 0.21% citric acid (90-95°C), followed by an incubation in 1% NaOH (w/v) and 2% H 2 O 2 (w/v) for 20 min, 0.3% glycine (w/v) for 10 min, and 0.03% SDS (w/v) for 10 min at room temperature. All brain sections were blocked with 10% normal goat serum (Vector Labs) and 0.3% Triton X-100 (Sigma) in PBS for 1 h. Mouse brain sections were incubated with rat anti-GFAP (1:250; #130300; Invitrogen), rabbit anti-GFAP (1:500; #Z0334, Dako), mouse anti-Aβ (IC16; 1:250; provided by Dr. C. Pietrzik, Mainz University), rabbit anti-pStat3 (1:500; #9145S, Cell Signaling), rabbit anti-Iba1 (1:250; 019-19741, Wako), rabbit anti-Stat3 (1:500; #12640, Cell Signaling), rabbit anti-RFP (1:300; #AB234, Evrogen), rat anti-LAMP1 (1:750; #121602, BioLegend), mouse anti-S100β (1:250; #S2532, Sigma), rat anti-C3d (1:250; A0063, Dako), rabbit anti-Ki67 (1:500; RM9106, Thermo Scientific), mouse anti-NeuN (1:100; MAB377, Millipore), rabbit anti-NG2 (1:250; AB5320, Millipore), and rabbit anti-Olig2 (1:500; A...
Mice were sacrificed, and one hemisphere was transferred to liquid nitrogen and stored at -80°C. Protein extraction was performed by homogenization in PBS (pH 7.4) with 1% phosphatase inhibitor and 1% protease inhibitor cocktails (Thermo Fisher) using a ceramic bead homogenizer (Precellys, VWR). Homogenates were extracted in RIPA buffer (in mM: 49.96 Tris, pH 7.2, 149.6 NaCl, 25.6 NP40, 24 NaDOC, 6.8 SDS) and centrifuged for 30 min at 100,000 g. The pellet containing insoluble Aβ was solubilized in SDS buffer (in mM: 69.2 SDS, 24.98 Tris, pH 7.5). Protein concentration was measured using a BCA Protein Assay Kit (Thermo Fisher) with a FLUOstar Omega Reader (BMG). Samples were solubilized in 4 × LDS buffer (Life Technologies), boiled for 10 min at 95°C, centrifuged for 5 min at 20,000 g at 4°C, and loaded on 4-12% NuPAGE Novex Bis-Tris Midi-gels (Life Technologies) for protein gel electrophoresis. SeeBlue Plus2 pre-stained protein standard (Thermo Fisher) was used to determine molecular weights. Following electrophoresis, the samples were transferred to nitrocellulose membranes (0.2 µm; Bio...
All immunohistochemical data points represent mean values of 5-10 brain sections per mouse. Confocal images were imported into ImageJ, converted to 8-bit images, and smoothed using a Gaussian filter. After contrast enhancement (0.4% saturated pixels), images were binarized using ImageJ (plaque stainings, automated MaxEntropy algorithm; GFAP and Iba1, a threshold defined as the mean background intensity plus the SD of background intensity multiplied by 2 was used). Astrocyte, microglia, and plaque area coverage as well as the number and size of plaques and LAMP1-positive dystrophic dendrites per area were quantified using ImageJ. The plaque area was subtracted from the area covered by dystrophic neurites before data analysis. Analysis of glial peri-plaque morphology was determined as described (Reichenbach et al, ). Briefly, following background removal, 3D particles were reconstructed using a Flood-Filler algorithm for each channel. Glial particles were considered as peri-plaque when they contained ≥ 1 voxels with a distance ≤ 2r to the plaque center. Near-plaque glial particles were smoothed using a Gaussian...
Machine-readable layer
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