Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease methods
Aim. Evidence-backed execution summary for Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease methods from Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease.
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mouse
Subject model for the experiment.
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- confirm full cohort details in the source paper
Western blot
reagent used in the protocol.
- Use
- Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail...
Quantitative RT-PCR
reagent used in the protocol.
- Use
- Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). One microgram of RNA was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions. RNA extraction and reverse-transcription were performed as described previously [...
Immunohistochemistry
reagent used in the protocol.
- Use
- Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were...
Stereotaxic injection and drug administration
reagent used in the protocol.
- Use
- For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm)....
Measurement of iron indices and serum parameters
reagent used in the protocol.
- Use
- Quantitative measurement of tissue nonheme iron was performed using the method of Torrance and Bothwelld [, ]. The results are presented as micrograms of iron per gram wet weight of tissue. Serum iron concentrations were determined using a serum iron assay kit (Nanjing Jiancheng Bioengineering institute, Nanjing, C...
Measurement of GSH and MDA levels
reagent used in the protocol.
- Use
- Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and then decapitated, and the hippocampus was immediately removed. The fresh tissues of hippocampus were perfused with PBS containing heparin to remove blood and clots. After weighing the tissue, it was homogen...
Primary neuron culture and treatment
reagent used in the protocol.
- Use
- Mice primary hippocampal neurons were isolated as previously reported [ ]. Briefly, the hippocampi of the embryonic mice (C57) at E19 were collected and incubated with 0.25% trypsin in D-Hanks for 15 min. Then, neuronal plating medium containing DMEM/F12 with 10% FBS was added to the hippocampi, and then they...
Assessment of neuronal viability in neuronal cultures
reagent used in the protocol.
- Use
- Analysis of neuronal survival was measured with a CCK8 assay (YEASEN, Wuhan, China, Catalog 40710ES03) and PI staining (YEASEN, Wuhan, China, Catalog 40755ES64), according to the instructions of the manufacturer. Primary neuron cells were washed with the medium after treatment, and then CCK8 was added to the medium...
Western blot
Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail...
- Use
- Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail...
Immunohistochemistry
Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were...
- Use
- Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were...
Stereotaxic injection and drug administration
For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm)....
- Use
- For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm)....
Morris water maze
The Morris water maze was performed as previously described [ ]. Briefly, mice were trained for five consecutive days to find a platform hidden 1 cm under water using a stationary array of cues on the walls. A digital tracking device was connected to a computer and was used to track the movement of the mice in...
- Use
- The Morris water maze was performed as previously described [ ]. Briefly, mice were trained for five consecutive days to find a platform hidden 1 cm under water using a stationary array of cues on the walls. A digital tracking device was connected to a computer and was used to track the movement of the mice in...
Contextual and cue fear conditioning
The experimental device and recording software [FCT-100] were purchased from Tai Meng Technology Co., Ltd. (Chengdu, China). Before training, the mice were placed into a single chamber (33 × 33 × 35 cm) with a metal grid at the bottom for 5 min to adapt to the novel envi...
- Use
- The experimental device and recording software [FCT-100] were purchased from Tai Meng Technology Co., Ltd. (Chengdu, China). Before training, the mice were placed into a single chamber (33 × 33 × 35 cm) with a metal grid at the bottom for 5 min to adapt to the novel envi...
Open field test
The open field test chamber was a rectangular chamber (60 × 60 × 40 cm) composed of gray polyvinyl chloride. The center area was illuminated by 25-W halogen bulbs (200 cm above the field). The mice were gently placed in one corner of the testing chamber and were allowed...
- Use
- The open field test chamber was a rectangular chamber (60 × 60 × 40 cm) composed of gray polyvinyl chloride. The center area was illuminated by 25-W halogen bulbs (200 cm above the field). The mice were gently placed in one corner of the testing chamber and were allowed...
Magnetic resonance imaging (MRI)
All the MRI examinations were performed with a 3.0-T MRI system (Discovery W750, GE) with a small animal coil. The protocol included axial fast-spin echo (FSE) T2-weighted imaging and an axial T1-weighted examination. A transverse T2-weighted image was obtained using the FSE sequence (repetition time/echo time [TR/T...
- Use
- All the MRI examinations were performed with a 3.0-T MRI system (Discovery W750, GE) with a small animal coil. The protocol included axial fast-spin echo (FSE) T2-weighted imaging and an axial T1-weighted examination. A transverse T2-weighted image was obtained using the FSE sequence (repetition time/echo time [TR/T...
Assessment of neuronal viability in neuronal cultures
Analysis of neuronal survival was measured with a CCK8 assay (YEASEN, Wuhan, China, Catalog 40710ES03) and PI staining (YEASEN, Wuhan, China, Catalog 40755ES64), according to the instructions of the manufacturer. Primary neuron cells were washed with the medium after treatment, and then CCK8 was added to the medium...
- Use
- Analysis of neuronal survival was measured with a CCK8 assay (YEASEN, Wuhan, China, Catalog 40710ES03) and PI staining (YEASEN, Wuhan, China, Catalog 40755ES64), according to the instructions of the manufacturer. Primary neuron cells were washed with the medium after treatment, and then CCK8 was added to the medium...
Statistics and data collection
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data are shown as the mean ± SD/SEM of at least three independent experiments. Statistical significance was considered at * p < 0.05, ** p < 0.01, *** p < 0.001. Unpaired t-tests (two-tailed) were used for single comparisons, and two-way ANOVA was used for mult...
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Western blot
Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail (Roche) on ice and boiled for 10 min. Proteins (10-50 µg) were separated by 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with anti-rabbit or anti-mouse IgG conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 h at room temperature, and detected using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Primary antibodies used were: Slc40A1 (Fpn1, Novus Biologicals, CO, USA)...
Immunohistochemistry
Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were cut with a vibrate. For immunohistochemistry, the slices were incubated with Fpn primary antibody (Alphadiagnosis, San Antonio, TX, USA catalog MTP11-A, 1:100) for 48 h, probed with biotin-labeled secondary antibodies (1:10,000) for 1 h at 37 °C, and detected with the DAB Kit (Zsbio, Beijing, China, Catalog ZLI-9018). The images were observed under a microscope (Olympus BX60, Tokyo, Japan). DAB-enhanced Perl's staining was used to detect iron accumulation as previously described [ ]. Sections of brain tissue were washed with PBS and incubated in...
Stereotaxic injection and drug administration
For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm). Adeno-associated viruses (AAVs) (10 12 IU/ml, 2 µl) for Fpn overexpression or lentiviruses (10 9 IU/ml, 2 µl) for Fpn knockdown and the relative control viruses were bilaterally microinfused into the hippocampus via a cannula connected to a Hamilton microsyringe (Reno, NV, USA). We injected the AAV packaged full length murine Fpn cDNA into the hippocampus of APPswe/PS1dE9 mice at 9 months old. 3 months later, behavioral tests were performed in these mice at 12 months old. The infusion rate was 0.2 µl/min, and the cannula was le...
Primary neuron culture and treatment
Mice primary hippocampal neurons were isolated as previously reported [ ]. Briefly, the hippocampi of the embryonic mice (C57) at E19 were collected and incubated with 0.25% trypsin in D-Hanks for 15 min. Then, neuronal plating medium containing DMEM/F12 with 10% FBS was added to the hippocampi, and then they were centrifuged at 1000 × g for 5 min. The cells were triturated and plated onto a plastic culture dish and incubated at 37 °C with 5% CO 2. After 7 days in vitro, recombinant human Aβ 1-42 (Chinese Peptide Company, Hangzhou, China, Catalog AMYD-002) was resuspended in DMSO to 5 mM as described previously [ ], and 5 mM Aβ 1-42 was diluted with sterile (1 × ) PBS to 100 µM on the day before treatment. Then, Aβ 1-42 solution was incubated for 24 h at 4 ˚C. After in...
Deficiency of Fpn in excitatory neurons induced brain atrophy and cognitive impairment
We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and littermate controls (Fig. ). In Morris water maze tests, we found that the KO mice showed significantly worse learning performance during the training sessions (Fig. ). In the probe trial, the KO mice displayed reduced accuracy, prolonged latency in finding the target platform, and a shorter duration in the target quadrant (Fig. ). Additionally, Fpn fl/fl/NEXcre mice spent remarkably less freezing time in a contextual fear memory task but not in cue fear memory, indicating impaired hippocampus-related learning/memory (Fig. ). No significant differences were observed in sensitivity to electrical shocks between the two groups (Fig. ). To avoid the possible developmental deficits caused...
Measurement outputs
What raw and processed outputs should exist?
Fpn-floxed (Fpn fl/fl ) mice, which were described previously [,, ], were obtained from Dr. N.C. Andrews and transferred to the C57BL/6 (C57) background. NEX-Cre mice [ ] were...
- 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
Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM...
- 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
Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). One microgram of RNA was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Toyobo, Osaka, J...
- 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
For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of t...
- 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
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
Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Fpn-floxed (Fpn fl/fl ) mice, which were described previously [,, ], were obtained from Dr. N.C. Andrews and transferred to the C57BL/6 (C57) background. NEX-Cre mice [ ] were...; Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM...; Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). One microgram of RNA was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Toyobo, Osaka, J...; For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of t....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). One microgram of RNA was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Toyobo, Osaka, J...; Data are shown as the mean ± SD/SEM of at least three independent experiments. Statistical significance was considered at * p < 0.05, ** p <...; To determine the precise role of Fpn in brain iron metabolism and cognitive impairment, we generated conditional knockout mice of Fpn (Fpn fl/fl/NEXcre ) by crossing the Fpn fl/...; We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and li...
from paperReporting output
Report representative outputs alongside summary comparisons for Fpn-floxed (Fpn fl/fl ) mice, which were described previously [,, ], were obtained from Dr. N.C. Andrews and transferred to the C57BL/6 (C57) background. NEX-Cre mice [ ] were..., Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM..., Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). One microgram of RNA was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Toyobo, Osaka, J..., For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of t....
inferred from protocolStructured statistical methods
Total RNA was extracted by TRIzol reagent (Invitrogen, CA, USA). One microgram of RNA was reverse transcribed into cDNA using a first strand cDNA synthesis kit (Toyobo, Osaka, J...; Data are shown as the mean ± SD/SEM of at least three independent experiments. Statistical significance was considered at * p < 0.05, ** p <...; To determine the precise role of Fpn in brain iron metabolism and cognitive impairment, we generated conditional knockout mice of Fpn (Fpn fl/fl/NEXcre ) by crossing the Fpn fl/...; We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and li...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail (Roche) on ice and boiled for 10 min. Proteins (10-50 µg) were separated by 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with anti-rabbit or anti-mouse IgG conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 h at room temperature, and detected using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Primary antibodies used were: Slc40A1 (Fpn1, Novus Biologicals, CO, USA); Slc40A1 (Fpn1, Alphadiagnosis, San Antonio, TX, USA); FTH (FTH1, ferritin heavy chain, Cell Signaling Technology, Danvers, MA, USA), Gpx4; actin (Proteintech, Wuhan, China). Detailed information about the antibodies is available in Supplementary Table.
Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were cut with a vibrate. For immunohistochemistry, the slices were incubated with Fpn primary antibody (Alphadiagnosis, San Antonio, TX, USA catalog MTP11-A, 1:100) for 48 h, probed with biotin-labeled secondary antibodies (1:10,000) for 1 h at 37 °C, and detected with the DAB Kit (Zsbio, Beijing, China, Catalog ZLI-9018). The images were observed under a microscope (Olympus BX60, Tokyo, Japan). DAB-enhanced Perl's staining was used to detect iron accumulation as previously described [ ]. Sections of brain tissue were washed with PBS and incubated in freshly prepared Perls solution (5% potassium ferrocyanide [Sigma-Aldrich, St. Louis, MO, USA]/10% hydrochloric acid) for 1 h, endogenous peroxidase activity was quenched for 20 min at room temperature in 0.3% H 2 O 2 in methanol, followed by washing with PBS five times. Then, DAB incubation...
For stereotaxic injection, mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg). Holes were drilled above the DG field of the hippocampus (bregma: anterior/posterior -2.0 mm, medial/lateral ±1.2 mm, and dorsal/ventral -2.2 mm). Adeno-associated viruses (AAVs) (10 12 IU/ml, 2 µl) for Fpn overexpression or lentiviruses (10 9 IU/ml, 2 µl) for Fpn knockdown and the relative control viruses were bilaterally microinfused into the hippocampus via a cannula connected to a Hamilton microsyringe (Reno, NV, USA). We injected the AAV packaged full length murine Fpn cDNA into the hippocampus of APPswe/PS1dE9 mice at 9 months old. 3 months later, behavioral tests were performed in these mice at 12 months old. The infusion rate was 0.2 µl/min, and the cannula was left in place for 10 min following completion of the infusion. AAVs for Fpn overexpression were purchased from Neuron Biotech (Shanghai, China), and lentivirus for Fpn short hairpin RNA was purchased from Genechem Co, Ltd. (Shanghai, China). Oligomeric Aβ 1-42 was injected into the mo...
Mice primary hippocampal neurons were isolated as previously reported [ ]. Briefly, the hippocampi of the embryonic mice (C57) at E19 were collected and incubated with 0.25% trypsin in D-Hanks for 15 min. Then, neuronal plating medium containing DMEM/F12 with 10% FBS was added to the hippocampi, and then they were centrifuged at 1000 × g for 5 min. The cells were triturated and plated onto a plastic culture dish and incubated at 37 °C with 5% CO 2. After 7 days in vitro, recombinant human Aβ 1-42 (Chinese Peptide Company, Hangzhou, China, Catalog AMYD-002) was resuspended in DMSO to 5 mM as described previously [ ], and 5 mM Aβ 1-42 was diluted with sterile (1 × ) PBS to 100 µM on the day before treatment. Then, Aβ 1-42 solution was incubated for 24 h at 4 ˚C. After incubation, the Aβ 1-42 solution was diluted into concentrations indicated for treatment with DMEM/F12. The neurons were treated with 10 µm/20 µm Aβ 1-42 as a lethal concentration for 24 h, and vehicle treatment was used for controls. Treatment with 100̴...
We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and littermate controls (Fig. ). In Morris water maze tests, we found that the KO mice showed significantly worse learning performance during the training sessions (Fig. ). In the probe trial, the KO mice displayed reduced accuracy, prolonged latency in finding the target platform, and a shorter duration in the target quadrant (Fig. ). Additionally, Fpn fl/fl/NEXcre mice spent remarkably less freezing time in a contextual fear memory task but not in cue fear memory, indicating impaired hippocampus-related learning/memory (Fig. ). No significant differences were observed in sensitivity to electrical shocks between the two groups (Fig. ). To avoid the possible developmental deficits caused by the genetic deletion of Fpn, we injected lentivirus packaged with a shRNA directed against Fpn into the hippocampus of 3-month-old C57 mice to knockdown the expression of Fpn in wild-type mice (Figs. and ). The behavior test was performed 2 months after the virus injection. Similar to the...
Machine-readable layer
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"name": "Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease methods",
"description": "Evidence-backed execution summary for Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease methods from Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease.",
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"text": "Mice were decapitated and the brain tissues of hippocampus, cortex and other parts were immediately removed. The tissues were homogenated with sample buffer (pH 7.6, 50 mM Tris-HCl, 10 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, and 0.2% bromophenol blue) and protease inhibitors cocktail (Roche) on ice and boiled for 10 min. Proteins (10-50 µg) were separated by 10% SDS-PAGE gel and transferred to nitrocellulose membranes (Merck Millipore, Burlington, MA, USA). The membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with anti-rabbit or anti-mouse IgG conjugated secondary antibodies (LI-COR, Lincoln, NE, USA) for 1 h at room temperature, and detected using the Odyssey Imaging System (LI-COR, Lincoln, NE, USA). Primary antibodies used were: Slc40A1 (Fpn1, Novus Biologicals, CO, USA)..."
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"text": "Mice were anesthetized using a mixture of ketamine (100 mg/kg) and dexmedetomidine (0.5 mg/kg) and immediately perfused with normal saline and 4% paraformaldehyde solution continuously. The brains were dissected and postfixed for 24 h at 4 °C, and 25-µm slices of brain tissues were cut with a vibrate. For immunohistochemistry, the slices were incubated with Fpn primary antibody (Alphadiagnosis, San Antonio, TX, USA catalog MTP11-A, 1:100) for 48 h, probed with biotin-labeled secondary antibodies (1:10,000) for 1 h at 37 °C, and detected with the DAB Kit (Zsbio, Beijing, China, Catalog ZLI-9018). The images were observed under a microscope (Olympus BX60, Tokyo, Japan). DAB-enhanced Perl's staining was used to detect iron accumulation as previously described [ ]. Sections of brain tissue were washed with PBS and incubated in..."
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"name": "Primary neuron culture and treatment",
"text": "Mice primary hippocampal neurons were isolated as previously reported [ ]. Briefly, the hippocampi of the embryonic mice (C57) at E19 were collected and incubated with 0.25% trypsin in D-Hanks for 15 min. Then, neuronal plating medium containing DMEM/F12 with 10% FBS was added to the hippocampi, and then they were centrifuged at 1000 × g for 5 min. The cells were triturated and plated onto a plastic culture dish and incubated at 37 °C with 5% CO 2. After 7 days in vitro, recombinant human Aβ 1-42 (Chinese Peptide Company, Hangzhou, China, Catalog AMYD-002) was resuspended in DMSO to 5 mM as described previously [ ], and 5 mM Aβ 1-42 was diluted with sterile (1 × ) PBS to 100 µM on the day before treatment. Then, Aβ 1-42 solution was incubated for 24 h at 4 ˚C. After in..."
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"name": "Deficiency of Fpn in excitatory neurons induced brain atrophy and cognitive impairment",
"text": "We next performed functional tests on learning and memory in Fpn fl/fl/NEXcre mice. No significant differences were found in basal locomotive behavior between the KO mice and littermate controls (Fig. ). In Morris water maze tests, we found that the KO mice showed significantly worse learning performance during the training sessions (Fig. ). In the probe trial, the KO mice displayed reduced accuracy, prolonged latency in finding the target platform, and a shorter duration in the target quadrant (Fig. ). Additionally, Fpn fl/fl/NEXcre mice spent remarkably less freezing time in a contextual fear memory task but not in cue fear memory, indicating impaired hippocampus-related learning/memory (Fig. ). No significant differences were observed in sensitivity to electrical shocks between the two groups (Fig. ). To avoid the possible developmental deficits caused..."
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"name": "Measurement of iron indices and serum parameters"
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"name": "Measurement of GSH and MDA levels"
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"headline": "Loss of ferroportin induces memory impairment by promoting ferroptosis in Alzheimer's disease",
"datePublished": "2021",
"author": [
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"@type": "Person",
"name": "Wen-Dai Bao"
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