Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein methods
Aim. Evidence-backed execution summary for Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein methods from Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Hematoxylin-eosin (H&E) staining
reagent used in the protocol.
- Use
- Lymph nodes were stained with hematoxylin solution for 5 min and washed with distilled water for 1 h, then placed in 70 and 90% alcohol for 10 min, respectively. The sections were stained with Eosin solution for 2-3 min, dehydrated with pure alcohol, transparented with xylene, and seale...
Stereotaxic injection of α-syn
reagent used in the protocol.
- Use
- AQP4 -/- mice and WT mice were given stereotaxic injection of soluble recombinant human α-syn. An injection cannula (27 gauge) was inserted stereotaxically into substantia nigra (SN) (anteroposterior: - 3.0 mm; mediolateral: ±1.3 mm; dorsoventral: - 4.2...
Section preparation
reagent used in the protocol.
- Use
- For section preparation, all mice were anesthetized with 4% chloral hydrate, and perfused with saline for 3 min followed by 4% PFA for 5 min. Brain tissues and Dclns were fixed in 4% PFA overnight after perfusion and dehydrated with 20% sucrose solution dissolved in 0.01 M PBS for 3 days. 30&...
Immunohistochemistry
reagent used in the protocol.
- Use
- Immunohistochemical staining was performed as previously described [ ]. The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MA...
Immunofluorescence
reagent used in the protocol.
- Use
- Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit ant...
LDclns exacerbates α-syn aggregation in A53T mouse brain
reagent used in the protocol.
- Use
- We also analyzed whether LDclns would affect clearance of other macromolecules from the brain. Results showed that protein levels of total Tau and PHF-1, one type of phosphorylated Tau, were higher in A53T-LDclns mice than those in WT-LDclns mice and A53T mice (all p < 0.05; Additional file:...
Brain homogenate preparation
reagent used in the protocol.
- Use
- Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 µl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min a...
Western blotting
reagent used in the protocol.
- Use
- Soluble and insoluble α-syn from ventral midbrain samples were quantified using a commercial ELISA kit (Abcam, Cat # ab210973). The assay procedure was carried out according to the manufacturer's instructions. Briefly, samples or standards were added to the 96-well plate and mixed with the antibody. After...
Open-field test
The experiment was used to assess autonomic motor ability in mice [ ]. Mice were placed in an open box (length: 60 cm, width: 60 cm; height: 25 cm) and allowed to move freely for 5 min. The total distance traveled was recorded by an open field software (Clever Sys Inc., VA, USA). After each t...
- Use
- The experiment was used to assess autonomic motor ability in mice [ ]. Mice were placed in an open box (length: 60 cm, width: 60 cm; height: 25 cm) and allowed to move freely for 5 min. The total distance traveled was recorded by an open field software (Clever Sys Inc., VA, USA). After each t...
Rotarod test
Rotarod test was used to detect the ability of motor coordination on a rotating rod (diameter 3.2 cm) [ ]. Mice were trained for 5 min, 3 times a day for 3 days. The speed started at 4 rpm and accelerated at 0.1 rpm/s. The test was performed three times per mouse, and the time of mouse...
- Use
- Rotarod test was used to detect the ability of motor coordination on a rotating rod (diameter 3.2 cm) [ ]. Mice were trained for 5 min, 3 times a day for 3 days. The speed started at 4 rpm and accelerated at 0.1 rpm/s. The test was performed three times per mouse, and the time of mouse...
Ligation of the deep cervical lymph nodes (LDclns)
The procedure of LDclns was performed according to published literature [ ]. Briefly, after anesthetized with 4% chloral hydrate, 18-week-old A53T mice and WT mice were fixed on a stereotaxic apparatus in a supine position, and longitudinally incised along the midline of the neck (approximately 1 cm). Under a...
- Use
- The procedure of LDclns was performed according to published literature [ ]. Briefly, after anesthetized with 4% chloral hydrate, 18-week-old A53T mice and WT mice were fixed on a stereotaxic apparatus in a supine position, and longitudinally incised along the midline of the neck (approximately 1 cm). Under a...
Intracisternal infusion of fluorescent tracer
Intracisternal infusion of fluorescent tracer was performed as previously described [ ]. Anesthetized mouse was fixed in a stereotaxic instrument, and the posterior atlanto-occipital membrane was exposed. Five microliter of Texas Red-dextran-3 (TR-d3, MW 3 kD; Invitrogen; Cat # D3328) at 0.5 mg ml -...
- Use
- Intracisternal infusion of fluorescent tracer was performed as previously described [ ]. Anesthetized mouse was fixed in a stereotaxic instrument, and the posterior atlanto-occipital membrane was exposed. Five microliter of Texas Red-dextran-3 (TR-d3, MW 3 kD; Invitrogen; Cat # D3328) at 0.5 mg ml -...
Cell counting and image analysis
Immunohistochemically stained sections were observed by an Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA) with an optical fractionator (Stereo Investigator 7, MBF bioscience, Williston, VT, USA). The numbers of TH-, GFAP- and Iba-1-positive cells in SN were assessed as described previously [ ]. Br...
- Use
- Immunohistochemically stained sections were observed by an Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA) with an optical fractionator (Stereo Investigator 7, MBF bioscience, Williston, VT, USA). The numbers of TH-, GFAP- and Iba-1-positive cells in SN were assessed as described previously [ ]. Br...
Cell counting and image analysis
The midbrain sections with immunofluorescence and CSF fluorescence tracer were photographed by a digital microscope (Leica Microsystems, Wetzlar, Germany). The image was analyzed using Image J and fluorescence signal of AQP4, α-syn and TR-d3 was measured respectively, by using the interest grayscale threshold a...
- Use
- The midbrain sections with immunofluorescence and CSF fluorescence tracer were photographed by a digital microscope (Leica Microsystems, Wetzlar, Germany). The image was analyzed using Image J and fluorescence signal of AQP4, α-syn and TR-d3 was measured respectively, by using the interest grayscale threshold a...
Western blotting
Soluble and insoluble α-syn from ventral midbrain samples were quantified using a commercial ELISA kit (Abcam, Cat # ab210973). The assay procedure was carried out according to the manufacturer's instructions. Briefly, samples or standards were added to the 96-well plate and mixed with the antibody. After...
- Use
- Soluble and insoluble α-syn from ventral midbrain samples were quantified using a commercial ELISA kit (Abcam, Cat # ab210973). The assay procedure was carried out according to the manufacturer's instructions. Briefly, samples or standards were added to the 96-well plate and mixed with the antibody. After...
Flow cytometry
Mice were perfused from the left cardiac ventricle with cold PBS for 10 min. Meninges were dissected with fine forceps and digested in RPMI-1640 medium (Sigma-Aldrich, Cat # R0883) + 1.4 U/ml Collagenase VIII (Sigma-Aldrich, Cat # C2139) + 1 mg/ml DNase1 (Sigma-Aldrich, Cat...
- Use
- Mice were perfused from the left cardiac ventricle with cold PBS for 10 min. Meninges were dissected with fine forceps and digested in RPMI-1640 medium (Sigma-Aldrich, Cat # R0883) + 1.4 U/ml Collagenase VIII (Sigma-Aldrich, Cat # C2139) + 1 mg/ml DNase1 (Sigma-Aldrich, Cat...
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Materials and methods
A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-fold higher than that in the controls as determined by quantitative PCR. Genotyped male mice were housed under standard conditions (room temperature 18 ~ 22 °C, humidity 30 ~ 50%, well-ventilated, a 12-h light-dark cycle). In addition, three-month old male AQP4 knockout (AQP4 -/- ) mice in a CD1 genetic background [ ] were used to evaluate the effect of Aqp4 gene deletion on removal of injected exogenous α-syn clearance from the brain parenchyma. All efforts were made to minimize and reduce number of animals used. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Immunohistochemistry
Immunohistochemical staining was performed as previously described [ ]. The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MAB360) and rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, Wako, Cat # 019-19,741). After rinsing with PBS, the sections were incubated with biotinylated goat anti-mouse or rabbit IgG (11000) for 1 h at room temperature and thereafter incubated with streptavidin-biotin-peroxidase complex (Elite ABC Kit, Vector Laboratories, Cat # PK-7200) and visualized with 2,2′-diaminodiphenyl.
Immunofluorescence
Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody.
Brain homogenate preparation
Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 µl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min at 16000 rpm [ ]. To analysis insoluble α-syn oligomeric protein, 5 volumes of Triton X-100 insoluble lysate were added to the precipitated protein obtained from centrifugation, sonication, boiling at 95 °C for 20 min, and centrifuged again for additional 10 min at room temperature [ ]. Protein concentration of the samples was determined by a bicinchoninic acid protein assay kit (Kevgen Biotech, Cat # ab6672). Bovine). Bovine serum albumin (1 mg/ml) was used as standard protein.
Western blotting
The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h [ ]. Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit an...
Measurement outputs
What raw and processed outputs should exist?
A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-...
- 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
AQP4 -/- mice and WT mice were given stereotaxic injection of soluble recombinant human α-syn. An injection cannula (27 gauge) was inserted stereotaxically into...
- 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
Immunohistochemically stained sections were observed by an Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA) with an optical fractionator (Stereo Investigator 7,...
- 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 midbrain sections with immunofluorescence and CSF fluorescence tracer were photographed by a digital microscope (Leica Microsystems, Wetzlar, Germany). The image was analyze...
- 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
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
All data were expressed as means ± SEM.
from paperScoring or quantification
Quantify the primary readouts for this experiment: A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-...; AQP4 -/- mice and WT mice were given stereotaxic injection of soluble recombinant human α-syn. An injection cannula (27 gauge) was inserted stereotaxically into...; Immunohistochemically stained sections were observed by an Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA) with an optical fractionator (Stereo Investigator 7,...; The midbrain sections with immunofluorescence and CSF fluorescence tracer were photographed by a digital microscope (Leica Microsystems, Wetzlar, Germany). The image was analyze....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
All data were expressed as means ± SEM. Statistical analyses were performed with Prism 6 (Graph-Pad, La Jolla, CA, USA). Data were analyzed by two-way analysis of variance...; Based on impaired glymphatic solute transport, we investigated whether LDclns improved aggregation of endogenous α-syn in the brain interstitium of WT and A53T mice. There...; Previous literature reported that autophagy participates in intracellular degradation of α-syn and is impaired in A53T mice [, ]. We determined whether LDclns aggravated a...; Previous studies showed that excessive aggregation of α-syn induces glial activation, subsequently causing release of inflammatory cytokines, and eventually intensifying de...
from paperReporting output
Report representative outputs alongside summary comparisons for A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-..., AQP4 -/- mice and WT mice were given stereotaxic injection of soluble recombinant human α-syn. An injection cannula (27 gauge) was inserted stereotaxically into..., Immunohistochemically stained sections were observed by an Olympus BX52 microscope (Olympus America Inc., Melville, NY, USA) with an optical fractionator (Stereo Investigator 7,..., The midbrain sections with immunofluorescence and CSF fluorescence tracer were photographed by a digital microscope (Leica Microsystems, Wetzlar, Germany). The image was analyze....
inferred from protocolStructured statistical methods
All data were expressed as means ± SEM. Statistical analyses were performed with Prism 6 (Graph-Pad, La Jolla, CA, USA). Data were analyzed by two-way analysis of variance...; Based on impaired glymphatic solute transport, we investigated whether LDclns improved aggregation of endogenous α-syn in the brain interstitium of WT and A53T mice. There...; Previous literature reported that autophagy participates in intracellular degradation of α-syn and is impaired in A53T mice [, ]. We determined whether LDclns aggravated a...; Previous studies showed that excessive aggregation of α-syn induces glial activation, subsequently causing release of inflammatory cytokines, and eventually intensifying de...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (5)
A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-fold higher than that in the controls as determined by quantitative PCR. Genotyped male mice were housed under standard conditions (room temperature 18 ~ 22 °C, humidity 30 ~ 50%, well-ventilated, a 12-h light-dark cycle). In addition, three-month old male AQP4 knockout (AQP4 -/- ) mice in a CD1 genetic background [ ] were used to evaluate the effect of Aqp4 gene deletion on removal of injected exogenous α-syn clearance from the brain parenchyma. All efforts were made to minimize and reduce number of animals used. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.
Immunohistochemical staining was performed as previously described [ ]. The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MAB360) and rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, Wako, Cat # 019-19,741). After rinsing with PBS, the sections were incubated with biotinylated goat anti-mouse or rabbit IgG (11000) for 1 h at room temperature and thereafter incubated with streptavidin-biotin-peroxidase complex (Elite ABC Kit, Vector Laboratories, Cat # PK-7200) and visualized with 2,2′-diaminodiphenyl.
Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody.
Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 µl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min at 16000 rpm [ ]. To analysis insoluble α-syn oligomeric protein, 5 volumes of Triton X-100 insoluble lysate were added to the precipitated protein obtained from centrifugation, sonication, boiling at 95 °C for 20 min, and centrifuged again for additional 10 min at room temperature [ ]. Protein concentration of the samples was determined by a bicinchoninic acid protein assay kit (Kevgen Biotech, Cat # ab6672). Bovine). Bovine serum albumin (1 mg/ml) was used as standard protein.
The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h [ ]. Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit anti-phosphorylated Tau Ser396/Ser404 (PHF-1, 1:1000, Abcam, ab184951) then followed by overnight incubation at 4 °C. The blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h, and signals were detected by chemiluminescence...
Machine-readable layer
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"name": "Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein methods",
"description": "Evidence-backed execution summary for Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein methods from Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein.",
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"text": "A53T mice were constructed by Nanjing University [ ]. Age-matched wild-type (WT) mice were used as controls. The α-syn cDNA content of homozygous A53T mice was more than 2-fold higher than that in the controls as determined by quantitative PCR. Genotyped male mice were housed under standard conditions (room temperature 18 ~ 22 °C, humidity 30 ~ 50%, well-ventilated, a 12-h light-dark cycle). In addition, three-month old male AQP4 knockout (AQP4 -/- ) mice in a CD1 genetic background [ ] were used to evaluate the effect of Aqp4 gene deletion on removal of injected exogenous α-syn clearance from the brain parenchyma. All efforts were made to minimize and reduce number of animals used. The animal experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University."
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"text": "Immunohistochemical staining was performed as previously described [ ]. The sections were incubated overnight at 4 °C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH, 1:4000, Sigma-Aldrich, Cat # T1299), mouse anti-glial fibrillary acid protein (GFAP, 1:1000, Millipore, Cat # MAB360) and rabbit anti-ionized calcium binding adapter molecule 1 (Iba-1, 1:1000, Wako, Cat # 019-19,741). After rinsing with PBS, the sections were incubated with biotinylated goat anti-mouse or rabbit IgG (11000) for 1 h at room temperature and thereafter incubated with streptavidin-biotin-peroxidase complex (Elite ABC Kit, Vector Laboratories, Cat # PK-7200) and visualized with 2,2′-diaminodiphenyl."
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"text": "Brain slices were blocked for 1 h at room temperature with 5% bovine serum albumin, incubated with a mixture of mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787) and rabbit anti-AQP4 (1:300, Millipore, Cat # AB3594), rabbit anti-laminin (1:300, Sigma-Aldrich, Cat # L9393) or rabbit anti-TH (1:500; Sigma-Aldrich, Cat # SAB4502964), or mouse anti-CD31 (1:500, Sigma-Aldrich, Cat # P8590) and rabbit anti-AQP4. Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (Thermo Fisher Scientific, 1:1000, Cat # A-11034; Cat # A-11032) were incubated for 1 h at room temperature. Meninges were incubated with a monoclonal mouse anti-lymphatic vessel endothelial hyaluronan receptor 1 (lyve-1) antibody (1:1000, Abcam, Cat # ab33682) and thereafter incubated with Alexa Fluor 488 secondary antibody."
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"text": "Mice were anesthetized, and ventral midbrain was quickly dissected out, isolated, weighed and crushed. Radio immunoprecipitation assay (RIPA) protein lysate was then added at a mass/volume ratio of 1:10 (10 µl/1 mg) and lysed on ice for 30 min at 4 °C, and centrifuged 15 min at 16000 rpm [ ]. To analysis insoluble α-syn oligomeric protein, 5 volumes of Triton X-100 insoluble lysate were added to the precipitated protein obtained from centrifugation, sonication, boiling at 95 °C for 20 min, and centrifuged again for additional 10 min at room temperature [ ]. Protein concentration of the samples was determined by a bicinchoninic acid protein assay kit (Kevgen Biotech, Cat # ab6672). Bovine). Bovine serum albumin (1 mg/ml) was used as standard protein."
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"text": "The brain samples were transferred onto polyvinylidene fluoride membranes using a Bio-Rad miniprotein-III wet transfer unit, followed by blocking with 5% nonfat milk dissolved in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h [ ]. Membranes were then probed with the following primary antibodies: mouse anti-β-actin (1:1000, Sigma-Aldrich, Cat # A2228), mouse anti-TH (1:4000, Sigma-Aldrich Cat # T1299), mouse anti-α-syn (1:1000, BD Transduction Laboratories, Cat # 610787), goat anti-interleukin 1β (IL-1β; 1:1000, Sigma-Aldrich; Cat # I3767), rabbit anti-IL-6 (1:1000, Abcam, Cat # ab6672), rabbit anti-tumor necrosis factor-α (TNF-α; 1:800, Abcam, Cat # ab9739), rabbit anti-light chain 3B (LC3B, 1:1000, CST, Cat # 2775), rabbit anti-p62 (1:500, Abcam, Cat # ab56416), rabbit anti-tau (1:1000, Abcam, Cat # ab32057) or rabbit an..."
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"name": "Ligation of the deep cervical lymph nodes (LDclns)"
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"name": "LDclns exacerbates α-syn aggregation in A53T mouse brain"
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"headline": "Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein",
"datePublished": "2019",
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"identifier": "10.1186/s40035-019-0147-y"
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"name": "Blocking meningeal lymphatic drainage aggravates Parkinson's disease-like pathology in mice overexpressing mutated α-synuclein methods",
"item": "https://replicatescience.com/experiments/blocking-meningeal-lymphatic-drainage-aggravates-parkinson-s-disease-like-pathology-in-mice-overexpressing-mutated-945-synuclein-methods-wenyan-zou-pmc6396507/blocking-meningeal-lymphatic-drainage-aggravates-parkinson-s-disease-like-pathology-in-mice-overexpr-mlpgxg1j"
}
]
}
]