Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice methods
Aim. Evidence-backed execution summary for Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice methods from Neuroinflammation induced by lipopolysaccharide causes cognitive impairment in mice.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Immunofluorescence staining
reagent used in the protocol.
- Use
- To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in...
ELISA
reagent used in the protocol.
- Use
- The frozen brains were homogenized in 100 mg tissue/mL cold PBS. The samples were centrifuged at 12,000 × g for 15 min. The supernatant was collected for a protein assay using a BCA protein assay reagent kit (PIERCE, Milwaukee, WI). The serum and brain levels of IL-1β (eBioscience,...
Western blot analysis
reagent used in the protocol.
- Use
- The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ...
Expression of proinflammatory cytokines in LPS-induced mice
reagent used in the protocol.
- Use
- To investigate the proinflammatory reaction in the LPS-induced mice, we measured the expression levels of proinflammatory cytokines such as TNF-α, IL-1β, PGE 2 and NO, in the serum and brain homogenates. As shown in Fig., the expression levels of TNF-α and IL-1β in the serum and brain homo...
Expression of anti-inflammatory cytokines in LPS-induced mice
reagent used in the protocol.
- Use
- Subsequently, we investigated the expression of IL-4 and IL-10, which are two important anti-inflammatory cytokines, in the LPS-induced mice. The serum and brain homogenate expression levels of IL-4 and IL-10 in the mice undergoing LPS administration for 1 or 7 consecutive days were significantly decreased compared...
VIPER attenuates the cognitive impairment, proinflammatory cytokines and NF-κB activity in LPS-induced mice
reagent used in the protocol.
- Use
- Lysakova et al. have identified VIPER, an inhibitor peptide of TLR-4, could directly interact with the TLR-4 adaptor proteins MyD88 adaptor-like (Mal) and TRIF-related adaptor molecule (TRAM). In addition, it has been reported that VIPER abrogated LPS-induced NF-κB activation. Thus, we observed the effect of...
Morris water maze test
The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered o...
- Use
- The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered o...
Passive avoidance performance test
The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box fo...
- Use
- The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box fo...
Immunofluorescence staining
To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in...
- Use
- To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in...
Western blot analysis
The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ...
- Use
- The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ...
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Materials and Methods
C57BL/6J male mice (11-12 weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All animal experiments were approved and carried out according to the Animal Ethics Committee of Jinan University (CBNUA-436-12-02). All mice were housed in a room with automatically controlled temperature (21-25 °C), relative humidity (45-65%), and light-dark (12-12 h) cycles. The mice in each cage were divided into the following treatment groups for each of the five experiments: (I) i.p. saline group (control), (II) i.p. LPS (500 µg/kg) group, (III) i.p. LPS (750 µg/kg) group, (IV) i.c.v. saline group, and (V) i.c.v. LPS (12 µg) group. Each group consisted of ten male mice. The i.p. LPS injections were administered at doses of 500 or 750 µg/kg in saline for seven consecutive days, and the saline (0.9%...
Morris water maze test
The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2 °C. An escape platform (height: 14 cm, diameter: 4-5 cm) was submerged 1-1.5 cm below the surface of the water in a specific position. During the training trials, the mice were placed into the water in a random quadrant and allowed to locate the hidden platform for 60 s. Then, the mice were allowed to remain on the platform or were placed on the platform for 10 s at the end of each trial. The mice were trained with three trials per day for 7 days. After the t...
Passive avoidance performance test
The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box for the first time and the latency to enter the dark box after an electric shock was applied to the feet were observed and automatically recorded to probe the learning and memory abilities of the mice. The mice were placed in the illuminated compartment facing away from the dark compartment during the first three days of the training trials. Then, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before each daily test during the training phase (7 days). All mice were placed in the illuminated compartment, and upon moving completely into the dark...
Climbing pole test
All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60 cm in length and 1 cm in diameter with two layers of gauze on the outside. On the sixth day, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before the climbing pole test, and the following three times were recorded: the time it took for the mouse to climb down the upper half, the time it took for the mouse to climb down the lower half, and the time it took for the mouse to complete the total length of the pole. If the mouse completed the above three steps within 3 s, 6 s or longer than 6 s, the motor coordination score was 3 points, 2 points, or 1 point, respectively.
Immunofluorescence staining
To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in a 30% sucrose solution. After being transferred to a 30% sucrose solution, the brains were cut into 10-µm-thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea) and incubated at 37 °C overnight. After three 5-min washes with PBS (pH = 7.4) and permeabilization with Triton X-100 (0.3% in TBST) at room temperature, the brains sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and overnight at 4 °C with a 1:100 dilution of an antibody against microtubule-associated protein 2...
Western blot analysis
The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ® instructions (Thermo Scientific, Rockford, IL, USA) to obtain cytoplasmic and nuclear protein. An equal amount of protein was separated via SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h with a 5% skim milk solution and incubated overnight at 4 °C with specific antibodies (1:1000 dilution, Cell Signaling Technology Inc, MA, USA). Then, the membranes were incubated in a 1:15000 dilution of a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc, MA, USA) for 1 h at room temp...
Measurement outputs
What raw and processed outputs should exist?
The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purcha...
- 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
The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & M...
- 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
All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60...
- 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
The frozen brains were homogenized in 100 mg tissue/mL cold PBS. The samples were centrifuged at 12,000 × g for 15 min. The supernatant was collecte...
- 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
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
Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times.
from paperScoring or quantification
Quantify the primary readouts for this experiment: The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purcha...; The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & M...; All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60...; The frozen brains were homogenized in 100 mg tissue/mL cold PBS. The samples were centrifuged at 12,000 × g for 15 min. The supernatant was collecte....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times. The data are presented as the mean ± SEM and...; To verify the relationship between the LPS-induced activation of microglia and neuronal cell loss, we examined the induction of neuronal cell loss by LPS in the hippocampus, whi...; To investigate the proinflammatory reaction in the LPS-induced mice, we measured the expression levels of proinflammatory cytokines such as TNF-α, IL-1β, PGE 2 and NO,...; Subsequently, we investigated the expression of IL-4 and IL-10, which are two important anti-inflammatory cytokines, in the LPS-induced mice. The serum and brain homogenate expr...
from paperReporting output
Report representative outputs alongside summary comparisons for The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purcha..., The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & M..., All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60..., The frozen brains were homogenized in 100 mg tissue/mL cold PBS. The samples were centrifuged at 12,000 × g for 15 min. The supernatant was collecte....
inferred from protocolStructured statistical methods
Unless otherwise stated, all experiments were performed with triplicate samples and repeated at least three times. The data are presented as the mean ± SEM and...; To verify the relationship between the LPS-induced activation of microglia and neuronal cell loss, we examined the induction of neuronal cell loss by LPS in the hippocampus, whi...; To investigate the proinflammatory reaction in the LPS-induced mice, we measured the expression levels of proinflammatory cytokines such as TNF-α, IL-1β, PGE 2 and NO,...; Subsequently, we investigated the expression of IL-4 and IL-10, which are two important anti-inflammatory cytokines, in the LPS-induced mice. The serum and brain homogenate expr...
source structuredSource and audit
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Evidence quotes (6)
C57BL/6J male mice (11-12 weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All animal experiments were approved and carried out according to the Animal Ethics Committee of Jinan University (CBNUA-436-12-02). All mice were housed in a room with automatically controlled temperature (21-25 °C), relative humidity (45-65%), and light-dark (12-12 h) cycles. The mice in each cage were divided into the following treatment groups for each of the five experiments: (I) i.p. saline group (control), (II) i.p. LPS (500 µg/kg) group, (III) i.p. LPS (750 µg/kg) group, (IV) i.c.v. saline group, and (V) i.c.v. LPS (12 µg) group. Each group consisted of ten male mice. The i.p. LPS injections were administered at doses of 500 or 750 µg/kg in saline for seven consecutive days, and the saline (0.9% NaCl) control mice received saline injections each day of testing. The i.c.v. injections of 12 µg of LPS (in 3 µL of saline) and saline (control) injections were administered on one day using a microsyringe and stereotaxic coordinates (-2.6 mm dorsal/ventral, -1.5 m...
The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2 °C. An escape platform (height: 14 cm, diameter: 4-5 cm) was submerged 1-1.5 cm below the surface of the water in a specific position. During the training trials, the mice were placed into the water in a random quadrant and allowed to locate the hidden platform for 60 s. Then, the mice were allowed to remain on the platform or were placed on the platform for 10 s at the end of each trial. The mice were trained with three trials per day for 7 days. After the training, on the final day of testing, we administered LPS or saline 6 h before conducting the spatial probe test. The escape latency and escape distance displayed by each mouse were recorded by a computer.
The passive avoidance test (PAT) is widely accepted as a simple memory testing method. The PAT was performed using a "step-through" apparatus (ChengDu Technology & Market Co, LTD) composed of six reaction boxes, allowing for six mice to be tested simultaneously. Here, the latency to enter the dark box for the first time and the latency to enter the dark box after an electric shock was applied to the feet were observed and automatically recorded to probe the learning and memory abilities of the mice. The mice were placed in the illuminated compartment facing away from the dark compartment during the first three days of the training trials. Then, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before each daily test during the training phase (7 days). All mice were placed in the illuminated compartment, and upon moving completely into the dark compartment, they received an electric shock (39 V, 3-s duration).
All mice were submitted to the "Pole test" to measure their motor coordination. Briefly, the mice were subjected to five days of training on a pole that was 60 cm in length and 1 cm in diameter with two layers of gauze on the outside. On the sixth day, LPS (i.p. 500 µg/kg or 750 µg/kg) was injected 6 h before the climbing pole test, and the following three times were recorded: the time it took for the mouse to climb down the upper half, the time it took for the mouse to climb down the lower half, and the time it took for the mouse to complete the total length of the pole. If the mouse completed the above three steps within 3 s, 6 s or longer than 6 s, the motor coordination score was 3 points, 2 points, or 1 point, respectively.
To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in a 30% sucrose solution. After being transferred to a 30% sucrose solution, the brains were cut into 10-µm-thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea) and incubated at 37 °C overnight. After three 5-min washes with PBS (pH = 7.4) and permeabilization with Triton X-100 (0.3% in TBST) at room temperature, the brains sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and overnight at 4 °C with a 1:100 dilution of an antibody against microtubule-associated protein 2 (MAP-2, Millipore Corp, Billerica, MA, USA), 1:200 dilution of an antibody against ionized calcium-binding adapter protein 1 (IBA-1, Millipore Corp, Billerica, MA, USA) and 1:100 dilution of an antibody against amyloid beta 1-42 (Aβ 1-42, Abcam, Cambridge, UK). On the following day...
The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ® instructions (Thermo Scientific, Rockford, IL, USA) to obtain cytoplasmic and nuclear protein. An equal amount of protein was separated via SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h with a 5% skim milk solution and incubated overnight at 4 °C with specific antibodies (1:1000 dilution, Cell Signaling Technology Inc, MA, USA). Then, the membranes were incubated in a 1:15000 dilution of a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc, MA, USA) for 1 h at room temperature. The signals were measured with an enhanced chemiluminescence kit (ECL, Millipore, USA) using a gel imaging system (Millipore, Billerica, MA, USA), and the results were visualized using Quantity One software.
Machine-readable layer
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"text": "C57BL/6J male mice (11-12 weeks old) were purchased from the Guangdong Medical Laboratory Animal Center. All animal experiments were approved and carried out according to the Animal Ethics Committee of Jinan University (CBNUA-436-12-02). All mice were housed in a room with automatically controlled temperature (21-25 °C), relative humidity (45-65%), and light-dark (12-12 h) cycles. The mice in each cage were divided into the following treatment groups for each of the five experiments: (I) i.p. saline group (control), (II) i.p. LPS (500 µg/kg) group, (III) i.p. LPS (750 µg/kg) group, (IV) i.c.v. saline group, and (V) i.c.v. LPS (12 µg) group. Each group consisted of ten male mice. The i.p. LPS injections were administered at doses of 500 or 750 µg/kg in saline for seven consecutive days, and the saline (0.9%..."
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"name": "Morris water maze test",
"text": "The Morris water maze (MWM) test, which is a commonly accepted method of testing memory, was performed as described by Morris et al.. The MWM program and equipment were purchased from ChengDu Technology & Market Co, LTD. A circular pool (height: 35 cm, diameter: 120 cm) was filled with water rendered opaque with whole milk and maintained at 23 ± 2 °C. An escape platform (height: 14 cm, diameter: 4-5 cm) was submerged 1-1.5 cm below the surface of the water in a specific position. During the training trials, the mice were placed into the water in a random quadrant and allowed to locate the hidden platform for 60 s. Then, the mice were allowed to remain on the platform or were placed on the platform for 10 s at the end of each trial. The mice were trained with three trials per day for 7 days. After the t..."
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"text": "To measure the microglia activation and neuronal cell loss in the mouse hippocampus, the mice were perfused with normal saline, followed by 4% paraformaldehyde (PFA) in 0.1 M sodium phosphate buffer (PBS), pH = 7.4. The cerebral tissues were removed, incubated overnight in fixatives, and stored in a 30% sucrose solution. After being transferred to a 30% sucrose solution, the brains were cut into 10-µm-thick sections using a cryostat microtome (Leica CM 1850; Leica Microsystems, Seoul, Korea) and incubated at 37 °C overnight. After three 5-min washes with PBS (pH = 7.4) and permeabilization with Triton X-100 (0.3% in TBST) at room temperature, the brains sections were blocked for 1 h in 1% bovine serum albumin (BSA) solution and overnight at 4 °C with a 1:100 dilution of an antibody against microtubule-associated protein 2..."
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"text": "The hippocampus tissues were homogenized in RIPA lysis buffer (Bioteke Co, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride and then centrifuged at 12,000 × g for 15 min at 4 °C. The cytoplasmic and nuclear p65 detection were performed according to the NE-PER ® instructions (Thermo Scientific, Rockford, IL, USA) to obtain cytoplasmic and nuclear protein. An equal amount of protein was separated via SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 1 h with a 5% skim milk solution and incubated overnight at 4 °C with specific antibodies (1:1000 dilution, Cell Signaling Technology Inc, MA, USA). Then, the membranes were incubated in a 1:15000 dilution of a horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology Inc, MA, USA) for 1 h at room temp..."
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"identifier": "10.1038/s41598-019-42286-8"
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