Late-onset Parkinsonism in NFκB/c-Rel-deficient mice methods
Aim. Evidence-backed execution summary for Late-onset Parkinsonism in NFκB/c-Rel-deficient mice methods from Late-onset Parkinsonism in NFκB/c-Rel-deficient mice.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
L-DOPA treatment
reagent used in the protocol.
- Use
- l -DOPA ( l -3,4-dihydroxyphenylalanine methyl ester hydrochloride) and benserazide hydrochloride were purchased from Sigma Aldrich and freshly dissolved in a saline solution at a final injection volume of 5 ml/kg. Both l -DOPA (20 mg/kg) and benserazide hydrochloride (12.5 mg/kg) or vehicle (saline) were injected i...
Materials and methods
reagent used in the protocol.
- Use
- C57BL/6 mice carrying the c-Rel gene null mutation (c-rel -/- ) were originally generated by inserting the neomycin cassette into the fifth exon of the c-Rel gene ( ). The c-rel -/- mice were backcrossed to C57BL/6 J mice for nine generations before being used in this study. The genotypes wer...
Immunohistochemistry
reagent used in the protocol.
- Use
- 3,3′-Diaminobenzidene immunostaining was performed on free-floating sections (30 µm) using primary antibodies: rabbit polyclonal anti-tyrosine hydroxylase (1:400 Millipore); polyclonal anti-choline acetyl transferase (ChAT; 1:500 Chemicon); mouse monoclonal anti-neuronal nuclei antibody (NeuN; clone A60,...
Immunohistochemistry
reagent used in the protocol.
- Use
- The double tyrosine hydroxylase staining was performed by incubating the sections previously processed for α-synuclein immunoreactivity in the anti-tyrosine hydroxylase antibody and biotinylated secondary antibody (goat anti-rabbit 1:250 Dako) followed by the avidin-biotin-horseradish peroxidase com...
Immunohistochemistry
reagent used in the protocol.
- Use
- For ferric iron, a Prussian blue stain with diaminobenzidine enhancement was used ( ). Sections were incubated with 7% potassium ferrocyanide (Perls reagent) in 3% HCl for 1 h at 37°C followed by distilled water and 0.015% H 2 O 2 -activated 0.75 mg/ml diaminobenzidine in 0.1 M PBS for 10 min.
Immunohistochemistry
reagent used in the protocol.
- Use
- Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit...
α-Synuclein extraction
reagent used in the protocol.
- Use
- Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS + ) buffer (Tris-HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N -ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice. The homogenate was centrifuged at 120 000 g for 30 min at 4°C. Th...
Immunoblot analysis
reagent used in the protocol.
- Use
- To analyse the tissue level of soluble α-synuclein, supernatants S1, S2 and S3 were loaded into SDS-PAGE gels and analysed by western blot technique. S4 supernatants were analysed to detect insoluble α-synuclein. Briefly, protein extracts were diluted in loading buffer (Sigma) and boiled for 2 min be...
L-DOPA treatment
Locomotor activity was individually measured as described above. Briefly, the animals were singularly placed in a novel PhenoTyper cage and spontaneous locomotor activity was video-recorded. Videos were then analysed by Ethovision XT software. For the CatWalk gait analysis, trained mice were treated with l -DOPA and...
- Use
- Locomotor activity was individually measured as described above. Briefly, the animals were singularly placed in a novel PhenoTyper cage and spontaneous locomotor activity was video-recorded. Videos were then analysed by Ethovision XT software. For the CatWalk gait analysis, trained mice were treated with l -DOPA and...
Immunohistochemistry
Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit...
- Use
- Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit...
Cell quantification
The number of tyrosine hydroxylase-, ChAT- and NeuN- immunopositive or Nissl-stained cells was stereologically estimated by double-blind cell counting in bright field microscopy using an optical fractionator method ( ). Neurons from the substantia nigra pars compacta, ventral tegmental area, basal forebrain and stri...
- Use
- The number of tyrosine hydroxylase-, ChAT- and NeuN- immunopositive or Nissl-stained cells was stereologically estimated by double-blind cell counting in bright field microscopy using an optical fractionator method ( ). Neurons from the substantia nigra pars compacta, ventral tegmental area, basal forebrain and stri...
Cell quantification
The optical density of striatal tyrosine hydroxylase-positive fibres was examined from digitized images using Image-Pro® Plus software (version 6.2, Media Cybernetics). Brains from four mice (three sections from each mouse) were analysed by examining an average of 10 fields per section.
- Use
- The optical density of striatal tyrosine hydroxylase-positive fibres was examined from digitized images using Image-Pro® Plus software (version 6.2, Media Cybernetics). Brains from four mice (three sections from each mouse) were analysed by examining an average of 10 fields per section.
Immunoblot analysis
DMT1 immunoreactivity was examined in total mesencephalic and striatal extracts by using a pan-DMT1 antibody (Santa Cruz Biotechnology). The levels of dopamine transporter were analysed in striatal extracts using the anti-dopamine transporter rat monoclonal antibody (Santa Cruz Biotechnology 1:200). Gel analysis was...
- Use
- DMT1 immunoreactivity was examined in total mesencephalic and striatal extracts by using a pan-DMT1 antibody (Santa Cruz Biotechnology). The levels of dopamine transporter were analysed in striatal extracts using the anti-dopamine transporter rat monoclonal antibody (Santa Cruz Biotechnology 1:200). Gel analysis was...
Behavioural studies
Spontaneous motility was assessed in a quiet isolated room. Mice were placed individually in plexiglass cages (length 47 cm, height 19 cm, width 27 cm) with a metal grid over the floor and equipped with infrared photocell emitters-detectors situated along the long axis of each cage (Opto-Varimex Mini; Columbus Instr...
- Use
- Spontaneous motility was assessed in a quiet isolated room. Mice were placed individually in plexiglass cages (length 47 cm, height 19 cm, width 27 cm) with a metal grid over the floor and equipped with infrared photocell emitters-detectors situated along the long axis of each cage (Opto-Varimex Mini; Columbus Instr...
CatWalk gait analysis
Gait parameters were evaluated using the CatWalk® 7.1 system (Noldus). All data analyses were performed with a pixel threshold value ≥25 arbitrary units. Mice were subjected to 3 days of training before the test. Mice were trained to cross the walkway (30 min/day) and were rewarded after every successful...
- Use
- Gait parameters were evaluated using the CatWalk® 7.1 system (Noldus). All data analyses were performed with a pixel threshold value ≥25 arbitrary units. Mice were subjected to 3 days of training before the test. Mice were trained to cross the walkway (30 min/day) and were rewarded after every successful...
Behavioural evaluation
Three separate groups of either c-rel -/- or wild-type mice at 4, 12 and 18 months of age were tested for their spontaneous motor behaviour. Motor activity was evaluated as total activity and locomotor activity during 60 min of observation in photobeam cages recording the number of photocell beam interru...
- Use
- Three separate groups of either c-rel -/- or wild-type mice at 4, 12 and 18 months of age were tested for their spontaneous motor behaviour. Motor activity was evaluated as total activity and locomotor activity during 60 min of observation in photobeam cages recording the number of photocell beam interru...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analysis was performed with the GraphPad Prism 4.0 program. Comparisons between two groups were performed using the two-tailed unpaired Student's t -test. Data were expressed as the mean ± SEM. Statistical significance was accepted at the 95% confidence level ( P < 0.05).
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L-DOPA treatment
l -DOPA ( l -3,4-dihydroxyphenylalanine methyl ester hydrochloride) and benserazide hydrochloride were purchased from Sigma Aldrich and freshly dissolved in a saline solution at a final injection volume of 5 ml/kg. Both l -DOPA (20 mg/kg) and benserazide hydrochloride (12.5 mg/kg) or vehicle (saline) were injected intraperitoneally into mice 1 h before the beginning of the behavioural tests.
Materials and methods
C57BL/6 mice carrying the c-Rel gene null mutation (c-rel -/- ) were originally generated by inserting the neomycin cassette into the fifth exon of the c-Rel gene ( ). The c-rel -/- mice were backcrossed to C57BL/6 J mice for nine generations before being used in this study. The genotypes were verified by PCR analysis (wild-type c-rel forward primer, 5′-AAGTGGGGTTACAGGTGCTCA-3′; wild-type c-rel reverse primer, 5′-TTGCCAATAGGCTTAGTCAAATA-3′; c-rel neomycin reverse primer, 5′-CTCTCGTGGGATCATTGTTTTTC-3′). PCR conditions were 94°C for 30 s, 57°C for 30 s, 72° for 30 s (30 cycles). The c-rel -/- and c-rel +/+ (wild-type) lines were continued by homozygous breeding. Animals were housed in standard cages and maintained under a 12h/12h light:dark cycle with food and water available ad libitum. Humidity was...
Immunohistochemistry
Mice were anaesthetized with chloral hydrate (400 mg/kg intraperitoneally) and transcardially perfused with PBS (SIGMA), 4% (w/v) ice-cold paraformaldehyde and 14% (w/v) ice-cold picric acid (SIGMA). Coronal slices (30 or 10 µm thickness) were cut to obtain serial sections of the following cerebral areas using bregma-based coordinates ( ): substantia nigra pars compacta (anterior-posterior -2.54 to -3.40 mm), ventral tegmental area (anterior-posterior -2.92 to -3.80 mm), medial septal area (anterior-posterior 1.18 to 0.38 mm), nucleus basalis magnocellularis (anterior-posterior -0.34 to -1.34 mm) and striatum (anterior-posterior 1.70 to -2.30 mm).
Immunohistochemistry
For ferric iron, a Prussian blue stain with diaminobenzidine enhancement was used ( ). Sections were incubated with 7% potassium ferrocyanide (Perls reagent) in 3% HCl for 1 h at 37°C followed by distilled water and 0.015% H 2 O 2 -activated 0.75 mg/ml diaminobenzidine in 0.1 M PBS for 10 min.
Immunohistochemistry
Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit anti-tyrosine hydroxylase (1:200 Chemicon) antibody overnight at 4°C followed by incubation in Alexa Fluor® 405-conjugated secondary antibodies (1:400 Jackson ImmunoResearch). For thioflavin S/α-synuclein double staining, sections were incubated with 0.3% KMnO 4 for 3-5 min, washed with water and incubated with a solution of 0.1% NaBH 4 for 5 min and then placed in a high-concentration PO 4 buffer (411 mM NaCl, 8.1 mM KCl, 30 mM NaHPO 4, 5.2 mM KH 2 PO 4 ) pH 7.2. After washing, thioflavin S (Sigma-Aldrich) and α-synuclein immunostaining was perf...
Cell quantification
The number of cholinergic neurons in the basal forebrain was determined by counting ChAT-positive cells in the medial septal area and in the nucleus basalis magnocellularis. For the striatum, NeuN-positive cells with identifiable nuclei were counted in three regions selected between lateral ±1.00 to ±2.00 mm and ventral ±2.00 to ±3.00 mm, (520 × 380 µm; left and right). Values are expressed as the mean ± SEM.
Quantification of CD11b- and glial fibrillary acidic protein-positive cells
Quantification of CD11b and GFAP immunostaining was carried out in brain slices sections processed on the same day in order to avoid any difference in staining intensity among animals. All pictures were captured within the same session in order to avoid any difference in any differences in lighting conditions. In each section, the entire left and right substantia nigra pars compacta were analysed, whereas for the striatum evaluation, one portion from the dorsolateral striatum (lateral from ±1.65 to ±2.15; ventral from -2.00 to -2.40) and one from the ventromedial striatum (lateral from ±1.25 to ±1.75; ventral from -1.75 to -2.15, 520 × 380 µm; left and right) were analysed. For each animal, three sections from the substantia nigra pars compacta (anterior-posterior -2.92 mm, -3.28 mm and -3.64 mm from bregma,...
α-Synuclein extraction
Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS + ) buffer (Tris-HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N -ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice. The homogenate was centrifuged at 120 000 g for 30 min at 4°C. The supernatant (S1) was saved, and the pellet was dissolved in TBS + buffer with 1% Triton™ X-100 and centrifuged at 120 000 g for 30 min at 4°C to obtain the second supernatant (S2). The procedure was repeated, the supernatant (S3) was retained and the pellet was dissolved in urea 8 M (SIGMA) with SDS 5% to obtain the supernatant S4.
Measurement outputs
What raw and processed outputs should exist?
Locomotor activity was individually measured as described above. Briefly, the animals were singularly placed in a novel PhenoTyper cage and spontaneous locomotor activity was vi...
- 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
Statistical analysis was performed with the GraphPad Prism 4.0 program. Comparisons between two groups were performed using the two-tailed unpaired Student's t -test. Data...
- 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 variations in monoamines and their metabolites were evaluated by a one-way ANOVA followed by Tukey's post hoc test.
- 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
Motor activity was evaluated using a one-way ANOVA followed by the Newman-Keuls post hoc test. Performance in the PhenoTyper cages was evaluated with a two-tailed unpaired Stude...
- 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 run-level gait data for each animal and preserve the timepoint or treatment labeling.
inferred from protocolPreprocessing / cleaning
Statistical analysis was performed with the GraphPad Prism 4.0 program.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Locomotor activity was individually measured as described above. Briefly, the animals were singularly placed in a novel PhenoTyper cage and spontaneous locomotor activity was vi...; Statistical analysis was performed with the GraphPad Prism 4.0 program. Comparisons between two groups were performed using the two-tailed unpaired Student's t -test. Data...; The variations in monoamines and their metabolites were evaluated by a one-way ANOVA followed by Tukey's post hoc test.; Motor activity was evaluated using a one-way ANOVA followed by the Newman-Keuls post hoc test. Performance in the PhenoTyper cages was evaluated with a two-tailed unpaired Stude....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Statistical analysis was performed with the GraphPad Prism 4.0 program. Comparisons between two groups were performed using the two-tailed unpaired Student's t -test. Data...; The variations in monoamines and their metabolites were evaluated by a one-way ANOVA followed by Tukey's post hoc test.; Motor activity was evaluated using a one-way ANOVA followed by the Newman-Keuls post hoc test. Performance in the PhenoTyper cages was evaluated with a two-tailed unpaired Stude...; With the aim to investigate other brain regions possibly undergoing age-related degeneration, we evaluated the survival of dopaminergic neurons in the ventral tegmental area and...
from paperReporting output
Report representative outputs alongside summary comparisons for Locomotor activity was individually measured as described above. Briefly, the animals were singularly placed in a novel PhenoTyper cage and spontaneous locomotor activity was vi..., Statistical analysis was performed with the GraphPad Prism 4.0 program. Comparisons between two groups were performed using the two-tailed unpaired Student's t -test. Data..., The variations in monoamines and their metabolites were evaluated by a one-way ANOVA followed by Tukey's post hoc test., Motor activity was evaluated using a one-way ANOVA followed by the Newman-Keuls post hoc test. Performance in the PhenoTyper cages was evaluated with a two-tailed unpaired Stude....
inferred from protocolStructured statistical methods
Statistical analysis was performed with the GraphPad Prism 4.0 program. Comparisons between two groups were performed using the two-tailed unpaired Student's t -test. Data...; The variations in monoamines and their metabolites were evaluated by a one-way ANOVA followed by Tukey's post hoc test.; Motor activity was evaluated using a one-way ANOVA followed by the Newman-Keuls post hoc test. Performance in the PhenoTyper cages was evaluated with a two-tailed unpaired Stude...; With the aim to investigate other brain regions possibly undergoing age-related degeneration, we evaluated the survival of dopaminergic neurons in the ventral tegmental area and...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
l -DOPA ( l -3,4-dihydroxyphenylalanine methyl ester hydrochloride) and benserazide hydrochloride were purchased from Sigma Aldrich and freshly dissolved in a saline solution at a final injection volume of 5 ml/kg. Both l -DOPA (20 mg/kg) and benserazide hydrochloride (12.5 mg/kg) or vehicle (saline) were injected intraperitoneally into mice 1 h before the beginning of the behavioural tests.
C57BL/6 mice carrying the c-Rel gene null mutation (c-rel -/- ) were originally generated by inserting the neomycin cassette into the fifth exon of the c-Rel gene ( ). The c-rel -/- mice were backcrossed to C57BL/6 J mice for nine generations before being used in this study. The genotypes were verified by PCR analysis (wild-type c-rel forward primer, 5′-AAGTGGGGTTACAGGTGCTCA-3′; wild-type c-rel reverse primer, 5′-TTGCCAATAGGCTTAGTCAAATA-3′; c-rel neomycin reverse primer, 5′-CTCTCGTGGGATCATTGTTTTTC-3′). PCR conditions were 94°C for 30 s, 57°C for 30 s, 72° for 30 s (30 cycles). The c-rel -/- and c-rel +/+ (wild-type) lines were continued by homozygous breeding. Animals were housed in standard cages and maintained under a 12h/12h light:dark cycle with food and water available ad libitum. Humidity was kept at a constant level, and the room temperature was maintained at 22-23°C. All animal experiments were authorized by the Italian Ministry of Health and by the University of Brescia Animal Care Committee in compliance with the Italian guidelines for animal care (DL 116/92) and the Euro...
Mice were anaesthetized with chloral hydrate (400 mg/kg intraperitoneally) and transcardially perfused with PBS (SIGMA), 4% (w/v) ice-cold paraformaldehyde and 14% (w/v) ice-cold picric acid (SIGMA). Coronal slices (30 or 10 µm thickness) were cut to obtain serial sections of the following cerebral areas using bregma-based coordinates ( ): substantia nigra pars compacta (anterior-posterior -2.54 to -3.40 mm), ventral tegmental area (anterior-posterior -2.92 to -3.80 mm), medial septal area (anterior-posterior 1.18 to 0.38 mm), nucleus basalis magnocellularis (anterior-posterior -0.34 to -1.34 mm) and striatum (anterior-posterior 1.70 to -2.30 mm).
For ferric iron, a Prussian blue stain with diaminobenzidine enhancement was used ( ). Sections were incubated with 7% potassium ferrocyanide (Perls reagent) in 3% HCl for 1 h at 37°C followed by distilled water and 0.015% H 2 O 2 -activated 0.75 mg/ml diaminobenzidine in 0.1 M PBS for 10 min.
Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit anti-tyrosine hydroxylase (1:200 Chemicon) antibody overnight at 4°C followed by incubation in Alexa Fluor® 405-conjugated secondary antibodies (1:400 Jackson ImmunoResearch). For thioflavin S/α-synuclein double staining, sections were incubated with 0.3% KMnO 4 for 3-5 min, washed with water and incubated with a solution of 0.1% NaBH 4 for 5 min and then placed in a high-concentration PO 4 buffer (411 mM NaCl, 8.1 mM KCl, 30 mM NaHPO 4, 5.2 mM KH 2 PO 4 ) pH 7.2. After washing, thioflavin S (Sigma-Aldrich) and α-synuclein immunostaining was performed and the sections examined with a Zeiss, LSM 510 META confocal microscope (Carl Zeiss), with the laser set at λ = 405-488-543 nm and the height of the section scanning = 1 mm. Images (512 × 512 pixels) were then re-constructed using LSM Image Examiner (Carl Zeiss) and Adob...
The number of cholinergic neurons in the basal forebrain was determined by counting ChAT-positive cells in the medial septal area and in the nucleus basalis magnocellularis. For the striatum, NeuN-positive cells with identifiable nuclei were counted in three regions selected between lateral ±1.00 to ±2.00 mm and ventral ±2.00 to ±3.00 mm, (520 × 380 µm; left and right). Values are expressed as the mean ± SEM.
Quantification of CD11b and GFAP immunostaining was carried out in brain slices sections processed on the same day in order to avoid any difference in staining intensity among animals. All pictures were captured within the same session in order to avoid any difference in any differences in lighting conditions. In each section, the entire left and right substantia nigra pars compacta were analysed, whereas for the striatum evaluation, one portion from the dorsolateral striatum (lateral from ±1.65 to ±2.15; ventral from -2.00 to -2.40) and one from the ventromedial striatum (lateral from ±1.25 to ±1.75; ventral from -1.75 to -2.15, 520 × 380 µm; left and right) were analysed. For each animal, three sections from the substantia nigra pars compacta (anterior-posterior -2.92 mm, -3.28 mm and -3.64 mm from bregma, according to the mouse brain atlas by ) and three sections from the striatum (anterior-posterior -1.10 mm, 0.74 mm and 0.38 mm from bregma) were analysed. CD11b immunostaining was evaluated with the Scion Image analysis program (Scion Corp.). Inside each frame, the area occupied by grey...
Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS + ) buffer (Tris-HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N -ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice. The homogenate was centrifuged at 120 000 g for 30 min at 4°C. The supernatant (S1) was saved, and the pellet was dissolved in TBS + buffer with 1% Triton™ X-100 and centrifuged at 120 000 g for 30 min at 4°C to obtain the second supernatant (S2). The procedure was repeated, the supernatant (S3) was retained and the pellet was dissolved in urea 8 M (SIGMA) with SDS 5% to obtain the supernatant S4.
Machine-readable layer
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"name": "Late-onset Parkinsonism in NFκB/c-Rel-deficient mice methods",
"description": "Evidence-backed execution summary for Late-onset Parkinsonism in NFκB/c-Rel-deficient mice methods from Late-onset Parkinsonism in NFκB/c-Rel-deficient mice.",
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"text": "C57BL/6 mice carrying the c-Rel gene null mutation (c-rel -/- ) were originally generated by inserting the neomycin cassette into the fifth exon of the c-Rel gene ( ). The c-rel -/- mice were backcrossed to C57BL/6 J mice for nine generations before being used in this study. The genotypes were verified by PCR analysis (wild-type c-rel forward primer, 5′-AAGTGGGGTTACAGGTGCTCA-3′; wild-type c-rel reverse primer, 5′-TTGCCAATAGGCTTAGTCAAATA-3′; c-rel neomycin reverse primer, 5′-CTCTCGTGGGATCATTGTTTTTC-3′). PCR conditions were 94°C for 30 s, 57°C for 30 s, 72° for 30 s (30 cycles). The c-rel -/- and c-rel +/+ (wild-type) lines were continued by homozygous breeding. Animals were housed in standard cages and maintained under a 12h/12h light:dark cycle with food and water available ad libitum. Humidity was..."
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"name": "Immunohistochemistry",
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"name": "Immunohistochemistry",
"text": "Double immunofluorescence staining was performed in sections (10 µm) incubated with α-synuclein antibody overnight at 4°C followed by a goat anti-mouse secondary antibody conjugated with Cy3 (1:400 Jackson ImmunoResearch) for 30 min at room temperature. Slices were then incubated with a primary rabbit anti-tyrosine hydroxylase (1:200 Chemicon) antibody overnight at 4°C followed by incubation in Alexa Fluor® 405-conjugated secondary antibodies (1:400 Jackson ImmunoResearch). For thioflavin S/α-synuclein double staining, sections were incubated with 0.3% KMnO 4 for 3-5 min, washed with water and incubated with a solution of 0.1% NaBH 4 for 5 min and then placed in a high-concentration PO 4 buffer (411 mM NaCl, 8.1 mM KCl, 30 mM NaHPO 4, 5.2 mM KH 2 PO 4 ) pH 7.2. After washing, thioflavin S (Sigma-Aldrich) and α-synuclein immunostaining was perf..."
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"name": "Cell quantification",
"text": "The number of cholinergic neurons in the basal forebrain was determined by counting ChAT-positive cells in the medial septal area and in the nucleus basalis magnocellularis. For the striatum, NeuN-positive cells with identifiable nuclei were counted in three regions selected between lateral ±1.00 to ±2.00 mm and ventral ±2.00 to ±3.00 mm, (520 × 380 µm; left and right). Values are expressed as the mean ± SEM."
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"name": "Quantification of CD11b- and glial fibrillary acidic protein-positive cells",
"text": "Quantification of CD11b and GFAP immunostaining was carried out in brain slices sections processed on the same day in order to avoid any difference in staining intensity among animals. All pictures were captured within the same session in order to avoid any difference in any differences in lighting conditions. In each section, the entire left and right substantia nigra pars compacta were analysed, whereas for the striatum evaluation, one portion from the dorsolateral striatum (lateral from ±1.65 to ±2.15; ventral from -2.00 to -2.40) and one from the ventromedial striatum (lateral from ±1.25 to ±1.75; ventral from -1.75 to -2.15, 520 × 380 µm; left and right) were analysed. For each animal, three sections from the substantia nigra pars compacta (anterior-posterior -2.92 mm, -3.28 mm and -3.64 mm from bregma,..."
},
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"@type": "HowToStep",
"position": 8,
"name": "α-Synuclein extraction",
"text": "Twenty milligrams of brain tissue were homogenized in five volumes of Tris-buffered saline plus (TBS + ) buffer (Tris-HCl 50 mM pH 7.4, NaCl 175 mM, EDTA 5 mM, PMSF 0.1 mM and N -ethylmaleimide 1 mM; Sigma) using a glass homogenizer on ice. The homogenate was centrifuged at 120 000 g for 30 min at 4°C. The supernatant (S1) was saved, and the pellet was dissolved in TBS + buffer with 1% Triton™ X-100 and centrifuged at 120 000 g for 30 min at 4°C to obtain the second supernatant (S2). The procedure was repeated, the supernatant (S3) was retained and the pellet was dissolved in urea 8 M (SIGMA) with SDS 5% to obtain the supernatant S4."
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