Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model methods
Aim. Evidence-backed execution summary for Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model methods from Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Stat3 deletion in astrocytes normalizes cerebral network activity, and a reduction in hyperactivity attenuates Stat3&...
reagent used in the protocol.
- Use
- To investigate a reciprocal scenario in which astroglial hyperactivity reduces Stat3 activation, we implanted osmotic minipumps into APP/PS1 mice and treated them with the network-normalizing P2Y1R inhibitor MRS2179 or vehicle for 6 weeks.
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
reagent used in the protocol.
- Use
- The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-Whitney test).
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
reagent used in the protocol.
- Use
- The swimming velocity was not affected by the treatment (Mann-Whitney test).
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
reagent used in the protocol.
- Use
- In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO), whereas APP/PS1 mice treated with vehicle spent equal times in the target and all other quadrants (* P < 0.05, Wilcoxon matched...
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
reagent used in the protocol.
- Use
- To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (* P < 0.05, MannR...
Stat3 regulates plaque-associated astrocytic and microglial complexity
reagent used in the protocol.
- Use
- Using an anti-Ki67 antibody as a marker for cellular proliferation, we detected Ki67-positive cells (arrows) in the hippocampal dentate gyrus as a positive control. However, no Ki67 signal was detected around plaques (marked by arrowheads) of either APP/PS1-Stat3KO or APP/PS1-Stat3WT mice, in...
Astrocyte Stat3 deletion induces a phenotypical switch in astrocytes
reagent used in the protocol.
- Use
- Confirming lower expression of the "A1" marker C3d, Western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. Immunohistochemistry using an antibody against C3d revealed that lower expression of C3d particularly occurred in peri-plaque reactive astrocytes (arrows indicat...
Stat3 deletion in astrocytes normalizes cerebral network activity, and a reduction in hyperactivity attenuates Stat3&...
reagent used in the protocol.
- Use
- However, we also considered a reciprocal scenario in which network dysregulation is not only a downstream effect, but also a cause of reactive astrogliosis, for example, by calcium-dependent signaling pathways (Kanemaru et al, ). Having previously shown that blockade of the P2Y1 purinoreceptor (P2Y1R) no...
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (* P < 0.05, MannR...
- Use
- To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium activity. Systemic treatment with the Stat3 inhibitor reduced the hyperactive phenotype of cortical neurons (* P < 0.05, MannR...
Stat3 deletion in reactive astrocytes protects against cognitive decline in APP/PS1 mice
Having found that astroglial Stat3 signaling strongly modulates the inflammatory environment around plaques, Aβ burden and clearance, neurite degeneration, and network function, we next determined its effects on the severity and progression of cognitive decline. To this end, we subjected APP/PS1-Stat3KO,...
- Use
- Having found that astroglial Stat3 signaling strongly modulates the inflammatory environment around plaques, Aβ burden and clearance, neurite degeneration, and network function, we next determined its effects on the severity and progression of cognitive decline. To this end, we subjected APP/PS1-Stat3KO,...
Stat3 deletion in reactive astrocytes protects against cognitive decline in APP/PS1 mice
A Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (* P < 0.05, two-way repe...
- Use
- A Spatial learning and memory were assessed in the Morris Water Maze paradigm. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on days 4 and 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (* P < 0.05, two-way repe...
Stat3 deletion in reactive astrocytes protects against cognitive decline in APP/PS1 mice
A Spatial learning and memory were assessed in the Morris Water Maze. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on day 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (* P < 0.05, two-way repeated-measu...
- Use
- A Spatial learning and memory were assessed in the Morris Water Maze. APP/PS1-Stat3KO mice showed faster latencies to reach the hidden platform compared with APP/PS1-Stat3WT on day 5, but were similar to WT-Stat3WT and WT-Stat3KO mice (* P < 0.05, two-way repeated-measu...
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
Finally, as a preclinical proof-of-concept study and to rule out effects other than those related to Stat3 deletion in our genetic model, we tested the efficacy of SH-4-54, a small-molecule blood-brain barrier-permeable Stat3 inhibitor that shows accumulation to sufficient a...
- Use
- Finally, as a preclinical proof-of-concept study and to rule out effects other than those related to Stat3 deletion in our genetic model, we tested the efficacy of SH-4-54, a small-molecule blood-brain barrier-permeable Stat3 inhibitor that shows accumulation to sufficient a...
A preclinical systemic Stat3 inhibitor confers protection from cognitive decline
A APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (* P &#...
- Use
- A APP/PS1 mice were systemically treated with SH-4-54 for 6 weeks and compared to age-matched APP/PS1 mice treated with vehicle. In the Morris Water Maze test, treatment with SH-4-54 led to significantly faster latencies to reach the hidden platform on the last training day (* P &#...
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 (anter...
- Use
- 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 (anter...
In vivo two-photon microscopy
OGB-1 AM (Life Technologies; solubilized in 20% Pluronic/80% DMSO and diluted to 1 mM with PBS) and SR101 (100 µM; Sigma) were co-injected into the cortex at a depth of 100-200 µm using a glass micropipette (tip diameter, 4-10 µm) connected to a pneumatic in...
- Use
- OGB-1 AM (Life Technologies; solubilized in 20% Pluronic/80% DMSO and diluted to 1 mM with PBS) and SR101 (100 µM; Sigma) were co-injected into the cortex at a depth of 100-200 µm using a glass micropipette (tip diameter, 4-10 µm) connected to a pneumatic in...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Sample size was predetermined based on a statistical power of 0.8 using G*Power 3 analysis software (Faul et al, ) and based on previous experience. Sample sizes were not altered during the course of the study. Data were excluded from the analysis if an animal died during or between experiments, or if it did n...
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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...
Measurement outputs
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The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Astrocytes in AD models express transcripts associated with a potentially neurotoxic "A1" phenotype, as opposed to a potentially neuroprotective "A2" phe...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A, B Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of "A1" markers Amigo2 and C3, whereas G...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Confirming lower expression of the "A1" marker C3d, Western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. Immunohistochemistry using...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-Whitney test).
from paperScoring or quantification
Quantify the primary readouts for this experiment: The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-...; Astrocytes in AD models express transcripts associated with a potentially neurotoxic "A1" phenotype, as opposed to a potentially neuroprotective "A2" phe...; A, B Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of "A1" markers Amigo2 and C3, whereas G...; Confirming lower expression of the "A1" marker C3d, Western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. Immunohistochemistry using....
from paperStatistical comparison
The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-...; The swimming velocity was not affected by the treatment (Mann-Whitney test).; In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO)...; To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium...
from paperReporting output
Report representative outputs alongside summary comparisons for The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-..., Astrocytes in AD models express transcripts associated with a potentially neurotoxic "A1" phenotype, as opposed to a potentially neuroprotective "A2" phe..., A, B Quantitative PCR from cortex of APP/PS1-Stat3KO compared to APP/PS1-Stat3WT mice revealed lower expression of "A1" markers Amigo2 and C3, whereas G..., Confirming lower expression of the "A1" marker C3d, Western blot analysis indicated lower protein levels of C3d in APP/PS1-Stat3KO. Immunohistochemistry using....
inferred from protocolStructured statistical methods
The area under the curve (AUC) for the latency to reach the hidden platform was also smaller in APP/PS1 mice treated with the Stat3 inhibitor (* P < 0.05, Mann-...; The swimming velocity was not affected by the treatment (Mann-Whitney test).; In the probe trial test, APP/PS1 mice treated with the Stat3 inhibitor spent significantly more time in the target quadrant (TQ) compared to the mean of all other quadrants (AO)...; To verify target engagement in the brain, the same mice assessed in the Morris Water Maze were anesthetized and imaged using in vivo two-photon microscopy of calcium...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
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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"text": "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."
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"text": "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..."
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"text": "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..."
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"text": "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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