Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway methods
Aim. Evidence-backed execution summary for Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway methods from Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Methods
reagent used in the protocol.
- Use
- A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could produce antidepressant effects. Behaviors tests were carried out to examine depressive, anxiety-like and cognitive behaviors including social interaction (SI) test, sucrose preference test (SPT), open field test (OFT),...
Materials and methods
reagent used in the protocol.
- Use
- Male C57BL/6J mice (aged 7-8 weeks) and retired male CD-1 mice (aged 16-20 weeks) were obtained from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle (lights on at 8:00 a...
Sucrose preference test (SPT)
reagent used in the protocol.
- Use
- Before the experiment, mice were habituated with two identical water bottles for 3 days. Each mouse was water and food-deprived for 24 h and then provided with 1% water and sucrose solution. After 12 h, the bottles were weighted and the sucrose preference was calculated as [sucrose water intake/(sucro...
Nissl staining
reagent used in the protocol.
- Use
- 10-µm coronal cryosections were stained with Nissl staining solution (Beyotime, C0117, Shanghai, China) for 5 min at 37 °C. Then samples were washed using 95% ethyl alcohol for 5 min and dried. Sections were then washed twice in xylene for 5 min. After being sealed with neutral bal...
Immunofluorescence and image analysis
reagent used in the protocol.
- Use
- Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-...
Transmission electron microscopy (TEM)
reagent used in the protocol.
- Use
- Mice were anesthetized and perfused with phosphate-buffered solution (PBS) followed by 4% paraformaldehyde. Small Hip and mPFC tissues (1 mm 3 ) were quickly dissected and post-fixed overnight at 4 °C using 2.5% glutaraldehyde. Tissues were embedded and cut into along the coronal plane at a thickness...
Measurement of MDA, SOD, GSH, GSH-PX and T-AOC
reagent used in the protocol.
- Use
- The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced glutathione (GSH), the glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) within Hip and mPFC tissues were measured with MDA activity assay kit (No. A003-1-2), SOD activity assay kit (No. A...
Western blotting (WB)
reagent used in the protocol.
- Use
- The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated wi...
Methods
A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could produce antidepressant effects. Behaviors tests were carried out to examine depressive, anxiety-like and cognitive behaviors including social interaction (SI) test, sucrose preference test (SPT), open field test (OFT),...
- Use
- A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could produce antidepressant effects. Behaviors tests were carried out to examine depressive, anxiety-like and cognitive behaviors including social interaction (SI) test, sucrose preference test (SPT), open field test (OFT),...
Materials and methods
In the first experiment, mice were randomly divided into three groups (10 each group): Control (CON) + Vehicle, CSDS + Vehicle and CSDS + EDA [10 mg/kg, intraperitoneally (i.p.)]. The experiment schedule is shown in Fig. A. After 10 day CSDS model, mice were admini...
- Use
- In the first experiment, mice were randomly divided into three groups (10 each group): Control (CON) + Vehicle, CSDS + Vehicle and CSDS + EDA [10 mg/kg, intraperitoneally (i.p.)]. The experiment schedule is shown in Fig. A. After 10 day CSDS model, mice were admini...
Chronic social defeat stress (CSDS)
SI test was composed of two stages. In the first stage, the experimental mice were allowed to explore an open field arena (44 cm × 44 cm × 30 cm) with an empty plastic box (10 cm × 7 cm × 18 cm) for 2.5 min. In the...
- Use
- SI test was composed of two stages. In the first stage, the experimental mice were allowed to explore an open field arena (44 cm × 44 cm × 30 cm) with an empty plastic box (10 cm × 7 cm × 18 cm) for 2.5 min. In the...
Open field test (OFT)
A plain, 44 cm × 44 cm × 30 cm open field arena was used to assess the locomotor activity and anxiety-like behavior [ ]. After a 30 s habituation, the total distance traveled, time spent in center arena (14.7 cm × 14.7 cm) was recorded...
- Use
- A plain, 44 cm × 44 cm × 30 cm open field arena was used to assess the locomotor activity and anxiety-like behavior [ ]. After a 30 s habituation, the total distance traveled, time spent in center arena (14.7 cm × 14.7 cm) was recorded...
Elevated plus maze (EPM)
The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Animals were placed in the EPM to explore for 5 min and tim...
- Use
- The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Animals were placed in the EPM to explore for 5 min and tim...
Novel object recognition (NOR)
The NOR test was performed according to previous protocols [ ]. Briefly, the test was performed in an open field arena (44 cm × 44 cm × 30 cm). Objects were fixed to the open field arena and had different shapes, sizes and textures. The NOR test consists of two s...
- Use
- The NOR test was performed according to previous protocols [ ]. Briefly, the test was performed in an open field arena (44 cm × 44 cm × 30 cm). Objects were fixed to the open field arena and had different shapes, sizes and textures. The NOR test consists of two s...
Nissl staining
10-µm coronal cryosections were stained with Nissl staining solution (Beyotime, C0117, Shanghai, China) for 5 min at 37 °C. Then samples were washed using 95% ethyl alcohol for 5 min and dried. Sections were then washed twice in xylene for 5 min. After being sealed with neutral bal...
- Use
- 10-µm coronal cryosections were stained with Nissl staining solution (Beyotime, C0117, Shanghai, China) for 5 min at 37 °C. Then samples were washed using 95% ethyl alcohol for 5 min and dried. Sections were then washed twice in xylene for 5 min. After being sealed with neutral bal...
Immunofluorescence and image analysis
Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-...
- Use
- Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data were presented as mean ± SEM and analyzed with GraphPad Prism 8.0 or the statistical program SPSS 17.0. The normality of data was assessed individually and non-parametric test were used when p < 0.05. Homogeneity of variance was be verified by Brown-Forsythe test, when p R...
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Materials and methods
Male C57BL/6J mice (aged 7-8 weeks) and retired male CD-1 mice (aged 16-20 weeks) were obtained from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle (lights on at 8:00 a.m.), 60 ± 5% humidity, and a temperature of 23 ± 1 °C with access to water and food freely. All experimental procedures were conducted in accordance with the Ethics Committee of Chongqing Medical University. EDA was purchased from Sigma-Aldrich (St. Louis, USA) and was dissolved in Vehicle (NaCl, 0.9%) at a dosage of 10 mg/kg. EX527 (a Sirt1 inhibitor) and ML385 (a Nrf2 inhibitor) were obtained from MedChemExpress (New Jersey, USA). The doses of these three drugs were determined according to previous studies [ - ].
Elevated plus maze (EPM)
The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Animals were placed in the EPM to explore for 5 min and time spent in closed and open arms was calculated.
Immunofluorescence and image analysis
Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 2 h at room temperature. After blocking, the sections were incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with the secondary antibodies for 2 h at room temperature. Primary antibodies include: Rabbit anti-Iba-1 (1:500, Wako, 019-19741), Goat anti-GFAP (1:500, Abcam, ab53554), Rabbit anti-TREM2 (1:150, Proteintech, 13483-1-AP), Rabbit anti-Connexin 30 (1:200, Abcam, ab200866), Rabbit anti-Connexin 43 (1:100, Thermo, 710700). The nuclei were counterstained with DAPI for 10 min. Fluorescent images were capt...
Measurement of MDA, SOD, GSH, GSH-PX and T-AOC
The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced glutathione (GSH), the glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) within Hip and mPFC tissues were measured with MDA activity assay kit (No. A003-1-2), SOD activity assay kit (No. A001-3-2) and GSH activity assay kit (No. A006-2-1), GSH-PX activity assay kit (No. A005-1-2) and T-AOC assay kit (S0121, Beyotime, China). MDA, SOD, GSH and GSH-PX kits were purchased from Jiancheng Inc. (Nanjing, China). In addition, it was necessary to pre-treat the brain tissues before measuring the above assay kits. Briefly, the tissues were weighted and PBS (pH 7.2-7.4, 0.01 mol/L) was added at a 1:9 proportion (MDA, SOD, GSH and GSH-PX kits) and a 1:5 proportion for T-AOC. Next, the samples were treated with tissue lyser (Jingxin, China), followed...
Western blotting (WB)
The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated with Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4 (1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), Rabbit anti-NF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse anti-GAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C, and secondary anti-mouse HRP-conjugated (1:10,000, Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-ra...
Enzyme-linked immunosorbent assay (ELISA)
The levels of TREM2, GFAP, IL-1β, IL-6 and TNF-α in the Hip and mPFC were measured by ELISA kits (Jianglai Biological, Shanghai, China). The tissues were weighed, and PBS was added at a 1:9 proportion and homogenized. Then, the homogenate was centrifuged at 5000 rpm for 15 min, and collecting the supernatant to measure. According to the instructions, 100 µl standard/sample was added and incubated for 1 h at 37 °C. Then, biotin antibody was added into each well and incubated for 1 h at 37 °C. The plate was then washed with washing buffer and streptavidin-HRP was added for 30 min at 37 °C. Next, TMB substrate was added to each well and kept away from light for 15 min at 37 °C after washing. Finally, stop solution was added and the optical density was measured at 450 nm.
Measurement outputs
What raw and processed outputs should exist?
A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could produce antidepressant effects. Behaviors tests were carried out to examine dep...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
The CSDS paradigm was conducted as described previously [ ]. Retired male CD-1 mice were screened for 3 consecutive days and the aggressors were selected according to the follow...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
A plain, 44 cm × 44 cm × 30 cm open field arena was used to assess the locomotor activity and anxiety-like behavior [ ]. After a...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × R...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Capture matched images from the relevant tissue region using the same acquisition settings across samples.
inferred from protocolPreprocessing / cleaning
In the first experiment, mice were randomly divided into three groups (10 each group): Control (CON) + Vehicle, CSDS + Vehicle and CSDS + EDA [10 mg/kg, intraperitoneally (i.p.)].
from paperScoring or quantification
Quantify the primary readouts for this experiment: A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could produce antidepressant effects. Behaviors tests were carried out to examine dep...; The CSDS paradigm was conducted as described previously [ ]. Retired male CD-1 mice were screened for 3 consecutive days and the aggressors were selected according to the follow...; A plain, 44 cm × 44 cm × 30 cm open field arena was used to assess the locomotor activity and anxiety-like behavior [ ]. After a...; The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × R....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
In the first experiment, mice were randomly divided into three groups (10 each group): Control (CON) + Vehicle, CSDS + Vehicle and CSDS + EDA...; Data were presented as mean ± SEM and analyzed with GraphPad Prism 8.0 or the statistical program SPSS 17.0. The normality of data was assessed individually and...; MDD was thought to be primarily triggered through neuronal dysfunction in the Hip and mPFC [ ]. Based on existing evidence, Nissl staining was employed to observe neuronal morph...; Neuroinflammation is tightly associated with the onset of depression [, ]. Given that microglia and astrocytes play an essential role in neuroinflammation and undergo abnormall...
from paperReporting output
Report representative outputs alongside summary comparisons for A chronic social defeat stress (CSDS) depression model was performed to explore whether EDA could produce antidepressant effects. Behaviors tests were carried out to examine dep..., The CSDS paradigm was conducted as described previously [ ]. Retired male CD-1 mice were screened for 3 consecutive days and the aggressors were selected according to the follow..., A plain, 44 cm × 44 cm × 30 cm open field arena was used to assess the locomotor activity and anxiety-like behavior [ ]. After a..., The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × R....
inferred from protocolStructured statistical methods
In the first experiment, mice were randomly divided into three groups (10 each group): Control (CON) + Vehicle, CSDS + Vehicle and CSDS + EDA...; Data were presented as mean ± SEM and analyzed with GraphPad Prism 8.0 or the statistical program SPSS 17.0. The normality of data was assessed individually and...; MDD was thought to be primarily triggered through neuronal dysfunction in the Hip and mPFC [ ]. Based on existing evidence, Nissl staining was employed to observe neuronal morph...; Neuroinflammation is tightly associated with the onset of depression [, ]. Given that microglia and astrocytes play an essential role in neuroinflammation and undergo abnormall...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (6)
Male C57BL/6J mice (aged 7-8 weeks) and retired male CD-1 mice (aged 16-20 weeks) were obtained from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle (lights on at 8:00 a.m.), 60 ± 5% humidity, and a temperature of 23 ± 1 °C with access to water and food freely. All experimental procedures were conducted in accordance with the Ethics Committee of Chongqing Medical University. EDA was purchased from Sigma-Aldrich (St. Louis, USA) and was dissolved in Vehicle (NaCl, 0.9%) at a dosage of 10 mg/kg. EX527 (a Sirt1 inhibitor) and ML385 (a Nrf2 inhibitor) were obtained from MedChemExpress (New Jersey, USA). The doses of these three drugs were determined according to previous studies [ - ].
The EPM test was used to measure anxiety-related behavior in rodents [ ]. The apparatus was composed of two closed arms (30 cm × 6 cm × 15 cm) and two open arms (30 cm × 6 cm). Animals were placed in the EPM to explore for 5 min and time spent in closed and open arms was calculated.
Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 2 h at room temperature. After blocking, the sections were incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with the secondary antibodies for 2 h at room temperature. Primary antibodies include: Rabbit anti-Iba-1 (1:500, Wako, 019-19741), Goat anti-GFAP (1:500, Abcam, ab53554), Rabbit anti-TREM2 (1:150, Proteintech, 13483-1-AP), Rabbit anti-Connexin 30 (1:200, Abcam, ab200866), Rabbit anti-Connexin 43 (1:100, Thermo, 710700). The nuclei were counterstained with DAPI for 10 min. Fluorescent images were captured with a fluorescence microscope (VS200; Olympus, Japan) and a confocal microscope (A1R, Nikon, Japan). The number of cells was counted manually containing the intact hippocampus (Hip) and medial prefrontal cortex (mPFC) from 3 mice in each group. Branch length and branch numb...
The activities of the antioxidant enzymes of malondialdehyde (MDA), the superoxide dismutase (SOD), reduced glutathione (GSH), the glutathione peroxidase (GSH-PX) and total antioxidant capacity (T-AOC) within Hip and mPFC tissues were measured with MDA activity assay kit (No. A003-1-2), SOD activity assay kit (No. A001-3-2) and GSH activity assay kit (No. A006-2-1), GSH-PX activity assay kit (No. A005-1-2) and T-AOC assay kit (S0121, Beyotime, China). MDA, SOD, GSH and GSH-PX kits were purchased from Jiancheng Inc. (Nanjing, China). In addition, it was necessary to pre-treat the brain tissues before measuring the above assay kits. Briefly, the tissues were weighted and PBS (pH 7.2-7.4, 0.01 mol/L) was added at a 1:9 proportion (MDA, SOD, GSH and GSH-PX kits) and a 1:5 proportion for T-AOC. Next, the samples were treated with tissue lyser (Jingxin, China), followed by centrifuging the homogenate at 12,000 rpm for 10 min at 4 °C, and collecting the supernatant to measure.
The Hip and mPFC tissues were dissociated by radio-immunoprecipitation assay solution containing protease inhibitors and phosphatase inhibitors. The proteins in the samples were run on 10% SDS polyacrylamide gel and then transferred to polyvinylidene fluoride membrane (Millipore, USA). The proteins were incubated with Rabbit anti-Sirt1 (1:1000, Abcam, ab189494), Rabbit anti-Nrf2 (1:1000, Proteintech, 16396-1-AP), Rabbit anti-HO-1 (1:1000, Proteintech, 10701-1-AP), Rabbit anti-Gpx4 (1:3000, Abcam, ab125066), Rabbit anti-TLR4 (1:1000, Abcam, ab217274), Rabbit anti-NF-κB p65 (phospho S536) (1:1000, Abcam, ab76302), Rabbit anti-NF-κB p65 (1:1000, Cell Signaling, 8242) and Mouse anti-GAPDH (1:10,000, Abcam, ab8245) overnight at 4 °C, and secondary anti-mouse HRP-conjugated (1:10,000, Bio-rad, 170-6516) or secondary anti-rabbit HRP-conjugated antibodies (1:10,000, Bio-rad, 170-6515) were incubated for 2 h at room temperature. The signals were visualized using ECL chemiluminescence reagent (Beyotime; US Everbright Inc.).
The levels of TREM2, GFAP, IL-1β, IL-6 and TNF-α in the Hip and mPFC were measured by ELISA kits (Jianglai Biological, Shanghai, China). The tissues were weighed, and PBS was added at a 1:9 proportion and homogenized. Then, the homogenate was centrifuged at 5000 rpm for 15 min, and collecting the supernatant to measure. According to the instructions, 100 µl standard/sample was added and incubated for 1 h at 37 °C. Then, biotin antibody was added into each well and incubated for 1 h at 37 °C. The plate was then washed with washing buffer and streptavidin-HRP was added for 30 min at 37 °C. Next, TMB substrate was added to each well and kept away from light for 15 min at 37 °C after washing. Finally, stop solution was added and the optical density was measured at 450 nm.
Machine-readable layer
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"name": "Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway methods",
"description": "Evidence-backed execution summary for Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway methods from Edaravone ameliorates depressive and anxiety-like behaviors via Sirt1/Nrf2/HO-1/Gpx4 pathway.",
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"text": "Male C57BL/6J mice (aged 7-8 weeks) and retired male CD-1 mice (aged 16-20 weeks) were obtained from the Experimental Animal Centre of Chongqing Medical University (Chongqing, China). The experimental animals were housed in cages under a 12 h light/12 h dark cycle (lights on at 8:00 a.m.), 60 ± 5% humidity, and a temperature of 23 ± 1 °C with access to water and food freely. All experimental procedures were conducted in accordance with the Ethics Committee of Chongqing Medical University. EDA was purchased from Sigma-Aldrich (St. Louis, USA) and was dissolved in Vehicle (NaCl, 0.9%) at a dosage of 10 mg/kg. EX527 (a Sirt1 inhibitor) and ML385 (a Nrf2 inhibitor) were obtained from MedChemExpress (New Jersey, USA). The doses of these three drugs were determined according to previous studies [ - ]."
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"text": "Immunofluorescence staining was performed on frozen coronal sections of mice brains. After post-fixation and concentration dehydration, the brains were cut into 20 µm thick sections. Then the slices were washed in PBS for 5 min three times, blocked with 5% bovine serum albumin (BSA) and 0.2% Triton X-100 for 2 h at room temperature. After blocking, the sections were incubated with primary antibodies overnight at 4 °C, and then sequentially incubated with the secondary antibodies for 2 h at room temperature. Primary antibodies include: Rabbit anti-Iba-1 (1:500, Wako, 019-19741), Goat anti-GFAP (1:500, Abcam, ab53554), Rabbit anti-TREM2 (1:150, Proteintech, 13483-1-AP), Rabbit anti-Connexin 30 (1:200, Abcam, ab200866), Rabbit anti-Connexin 43 (1:100, Thermo, 710700). The nuclei were counterstained with DAPI for 10 min. Fluorescent images were capt..."
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