Materials & Equipment Checklist
10 items1 from ConductScience
Gather these items before starting the experiment. Check off items as you prepare.
Equipment2
Materials7
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
Millipore • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
Sigma-Aldrich • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
not specified • not specified • not specified • not mentioned
Software1
not specified • not mentioned
1 item available from ConductScience
Estimated: $690.00
As an Amazon Associate, we earn from qualifying purchases. Product links help support this free resource.
Protocol Steps
1
Antibody selection and characterization
Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (6H7) and C-terminus of α-synuclein (8A5, 9E4) to determine which antibodies displayed the most specific binding to human α-synuclein. 9E4 was selected for its superior specificity.
not specifiednot specified
Note: 9E4 antibody displayed the most specificity and was chosen for the immunization study
View evidence from paper
“Initial immunoblot and immunohistochemical studies were conducted with a panel of antibodies directed at both N-terminus (NT) (6H7) and CT-α-syn (8A5, 9E4) to determine which of these antibodies displayed the most specific binding to human α-syn, of these antibodies, 9E4 displayed the most specificity”
2
Passive immunization - main study initiation
A total of 40 α-syn transgenic mice (6 months old, n=20 mice per group) received weekly intraperitoneal injections of 9E4 antibody or IgG1 control at 10 mg/kg dose. An additional group of non-transgenic mice (n=12 per group) treated with 9E4 antibody and IgG1 control was included as control.
6 monthsnot specified
Note: Injections were administered weekly via intraperitoneal route
View evidence from paper
“A total of 40 α-syn tg mice (6 m/o, n=20 mice per group) received weekly intraperitoneal (IP) injections (10 mg/kg) for 6 months with the CT-α-syn antibody (9E4) and IgG1 control. An additional group of non-tg mice treated with the 9E4 antibody (n=12) and the IgG1 control (n=12)”
3
Monthly blood collection and antibody titer monitoring
Mice were bled once a month throughout the 6-month immunization period. Antibody titers were monitored by enzyme-linked immunosorbent assay (ELISA).
Monthly for 6 monthsnot specified
Note: ELISA used to quantify antibody titers in serum
View evidence from paper
“Mice were bled once a month and antibody titers monitored by enzyme-linked immunosorbent assay (ELISA)”
4
Water maze behavioral testing
At the end of the 6-month immunization study, mice were tested for functional effects in the water maze to assess cognitive and motor performance.
not specifiednot specified
Note: Conducted at end of 6-month treatment period
View evidence from paper
“At the end of the studies, mice were tested for functional effects in the water maze”
5
Brain and tissue collection
Following behavioral testing, brains and peripheral tissues were removed and divided sagittally for neuropathological and protein analysis.
not specifiednot specified
Note: Right hemibrain and left hemibrain processed separately
View evidence from paper
“Brains and peripheral tissues were removed and divided sagittally”
6
Antibody concentration for FITC conjugation
The mouse monoclonal antibody 9E4 was concentrated using a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA) in preparation for FITC conjugation.
not specifiednot specified
Note: Concentration step performed before fluorescent labeling
View evidence from paper
“the mouse monoclonal antibody 9E4 was concentrated with a 10-kDa cutoff concentrator centrifuge tube (Millipore, Temecula, CA)”
7
FITC conjugation of 9E4 antibody
The concentrated 9E4 antibody was linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions.
not specifiednot specified
Note: Performed according to kit manufacturer's instructions
View evidence from paper
“linked to the Fluorescein isothiocyanate (FITC) molecule utilizing a FluoroTag FITC conjugation kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions”
8
Antibody trafficking study - 9E4-FITC injection
Non-transgenic (n=18) and α-synuclein transgenic mice (n=18), 6 months old, were injected intravenously with 9E4-FITC or non-immune control FITC-tagged IgG1 at a concentration of 1 mg/kg.
Single injectionnot specified
Note: Intravenous route used for this trafficking study
View evidence from paper
“non-tg (total n=18) and α-syn tg mice (total n=18) (6 m/o) were injected intravenously (IV) with the 9E4-FITC or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg”
9
Antibody trafficking study - tissue collection at multiple timepoints
Mice from the 9E4-FITC injection study were sacrificed at 3, 14, and 30 days after injection (n=3 per timepoint group). Cerebrospinal fluid (CSF) was collected from these mice.
3, 14, and 30 days post-injectionnot specified
Note: Three separate timepoint groups with n=3 per group
View evidence from paper
“Mice were sacrificed 3, 14 and 30 days after injection (n=3 per group). CSF from these mice was used to immunolabel cortical sections”
10
Blood-brain barrier control study - β-synuclein injection
As an additional control to monitor the passage of FITC-labeled antibodies across the blood-brain barrier, non-transgenic (n=8) and α-synuclein transgenic mice (n=8), 6 months old, were injected intravenously with FITC-labeled β-synuclein or non-immune control FITC-tagged IgG1 at a concentration of 1 mg/kg.
Single injectionnot specified
Note: β-synuclein used as control protein to assess BBB permeability
View evidence from paper
“non-tg (total n=8) and α-syn tg mice (total n=8) (6 m/o) were injected intravenously (IV) with the FITC-labeled β-syn or a non-immune control FITC tagged IgG1 at a concentration of 1 mg/kg”
11
Blood-brain barrier control study - tissue collection
Mice from the β-synuclein injection study were sacrificed at 14 days post-injection. Cerebrospinal fluid (CSF) was collected and used to immunolabel slides from FITC-tagged β-synuclein naive non-transgenic and α-synuclein transgenic mice.
14 days post-injectionnot specified
Note: Single timepoint collection at 14 days
View evidence from paper
“Mice were sacrified at 14 days post-injection. CSF from these mice was also used to immunolabel slides from FITC tagged β-syn niave non-tg and α-syn tg mice”
12
Right hemibrain post-fixation for neuropathology
The right hemibrain was post-fixed in phosphate-buffered 4% paraformaldehyde (PFA) at pH 7.4 at 4°C for 48 hours for neuropathological analysis.
48 hours4°C
Note: pH 7.4 phosphate buffer used
View evidence from paper
“the right hemibrain was post-fixed in phosphate-buffered 4% PFA (pH 7.4) at 4°C for 48 hours for neuropathological analysis”
13
Left hemibrain snap-freezing for protein analysis
The left hemibrain was snap-frozen and stored at -70°C for subsequent protein analysis.
not specified-70°C
Note: Snap-freezing preserves protein integrity for biochemical analysis
View evidence from paper
“the left hemibrain was snap-frozen and stored at −70°C for subsequent protein analysis”
Subjects / Specimens
- Species
- mouse
- Strain
- α-synuclein transgenic (Line D) and non-transgenic
- Age
- 6 months old
- Sex
- unknown
- Count
- 82
- Weight
- not specified
α-syn tg mice (n=20 per group for main study, n=18 for trafficking study, n=8 for β-syn control); non-tg mice (n=12 per group for main study, n=18 for trafficking study, n=8 for β-syn control)