A subscription to JoVE is required to view this content. Sign in or start your free trial.

Abstract

Source: Panattoni, G., et al. Exogenous Administration of Microsomes-associated Alpha-synuclein Aggregates to Primary Neurons As a Powerful Cell Model of Fibrils Formation. J. Vis. Exp. (2018)

This video demonstrates the immunostaining of neurons treated with alpha-synuclein (αS) aggregates. The neurons are first treated with αS aggregates. They are then fixed, permeabilized, and treated with a blocking solution. Primary antibodies are added, followed by a fluorescently labeled secondary antibodies and a nuclear stain. The sample is then mounted and observed under a microscope.

Protocol

1. Neurons Treatment

Note: The treatment has been performed at Division (DIV) 7. All the steps are carried on under a cell culture hood in sterile conditions. An example of cortical neuronal culture density at DIV 7 is illustrated in Figure 1.

1. Pool microsomes-associated αS aggregates obtained from the spinal cord of three different diseased Transgenic mice to have a 1 µg/µL solution, using the original homogenization buffer [composed of 250 mM sucrose, 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 mM KCl (Potassium Chloride), 1.5 mM MgCl₂ ( (Magnesium Chloride), 2 mM EDTA (Ethylenediaminetetraacetic acid), and 1x phosphatase/protease-inhibitors] for the dilution.
2. Take a neuron culture on poly-D-Lysine coverslips in 24 well plates containing medium (2% B27, 10x gentamicin, and 2 mM glutamine). Remove 1/3 of the medium and replace it gently with the fresh medium containing 2% B27, 1x gentamicin, and 2 mM glutamine.
3. Add 1 µg of pooled microsomes-associated alpha-synuclein (αS) aggregates to the cell medium. Return neurons to the incubator at 37 °C.
4. Every 3 days for 1 week, add 1/3 of fresh medium. Do not replace the medium. Just add it.
5. After 1 week of treatment, remove 1/3 of the medium and replace it gently with the fresh medium containing 2% B27, 10 µg/mL gentamicin, and 2 mM glutamine. Repeat every 3 days.
6. Fix neurons after 2 weeks of treatment (DIV 21).

2. Immunofluorescence

1. Fix neurons with 2% paraformaldehyde in Phosphate-Buffered Saline (PBS) 1x and 5% sucrose solution for 15 min at room temperature (RT) without shaking under a chemical fume hood.
2. Remove the fixing solution. Briefly wash with PBS 1x, 3 times.
   NOTE: Perform all the washing steps gently because primary neurons do not stick firmly to poly-lysine coverslips.
3. Permeabilize neurons with 0.3% of non-ionic surfactant in PBS 1x for 5 min at RT.
4. Briefly wash with PBS 1x, 3 times.
5. Incubate neurons with 3% Fetal Bovine Serum (FBS) in PBS 1x for 30 min at RT to block unspecific binding sites on an orbital shaker.
6. Incubate neurons with appropriate primary antibody dissolved in 3% FBS in PBS 1x, O/N at 4 °C on an orbital shaker.
   NOTE: syn303 (1:1,000), mouse αS (1:200), pser129-αS (1:1,000), and Tau (1:10,000) antibodies were used.
7. Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.
8. Incubate neurons with appropriate fluorescent secondary antibody dissolved in 3% FBS in PBS for 1 h at RT in the dark on an orbital shaker.
9. Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.
10. Stain neurons with DAPI (4′,6-diamidino-2-phenylindole) solution (0.1 µg/mL in PBS 1x) for 15 min at RT in the dark on an orbital shaker.
11. Remove the antibody solution and wash, briefly, with PBS 1x, 3 times.
12. Mount coverslips on a slide using an antifade mounting medium.

Representative Results

Figure 1. Cortical neuronal cultures. Representative image showing density at DIV 7 of cortical neuronal cultures. Images were taken with an inverted light microscope, 10X objective. Scale bar = 100 µm.

Materials

Sucrose	Sigma-Aldrich	84097-1KG
Hepes	Sigma-Aldrich	H0887-100ML	1M pH=7-7.6
EDTA	Sigma-Aldrich	0390-100ml	pH=8 0.5M
Magnesium chloride	Sigma-Aldrich	M8266-100G
Sodium carbonate	Sigma-Aldrich	S7795-500G
Sodium bicarbonate	Sigma-Aldrich	S5761-500G
Methanol	Sigma-Aldrich	322415-6X1L
KCl	Sigma-Aldrich	P9541-500G
cOmplete Mini	Roche	11836170001	protease inhibitor
PhosStop	Roche	4906837001	phosphatase inhibitor
BCA Protein Assay Kit	Euroclone	EMPO14500
Criterion TGX 4-20% Stain Free, 18 wells	Biorad	5678094
Supported Nitrocellulose membrane	Biorad	1620097	0.2 μm
Blotting-Grade Blocker	Biorad	1706404	Non-fat dry milk
SuperSignal West Pico Chemiluminescent Substrate	Termo Fisher Scientific	34077
Nitric acid	Sigma-Aldrich	1004411000	65%
Glass Coverslips	Termo Fisher Scientific	1014355118NR1	18 mm x
Poly-D-Lysine	Sigma-Aldrich	P7280
Hank's Balanced Salt Solution	Termo Fisher Scientific	14170-500 mL
Penicillin/Streptomycin	Termo Fisher Scientific	15140122	10,000 U/mL, 100 mL
Dulbecco's Modified Eagle's Medium	Termo Fisher Scientific	D5796-500 mL
Trypsin-EDTA	Termo Fisher Scientific	15400054	0.50%
B27 Supplement	Termo Fisher Scientific	17504044	50X
Glutamax	Termo Fisher Scientific	35050-038	100x
DNAse	Sigma-Aldrich	D5025
Fetal bovine serum	Euroclone	EC50182L
Glutamate	Sigma-Aldrich	1446600-1G
Gentamicin	Termo Fisher Scientific	15710	10 mg/ml
Neurobasal Medium	Termo Fisher Scientific	10888-022
Cytosine arabinoside (AraC)	Sigma-Aldrich	C3350000
VECTASHIELD antifade mounting medium	Vector Laboratories	H-1000
DAPI	Termo Fisher Scientific	62247
90 Ti rotor	Beckman	N/A	Ultracentrifuge rotor
Optima L-90K Ultracentrifuge	Beckman	N/A
Syn-1 antibody, clone 42	BD Biosciences	610786	anti-mouse WB: 1:5000
Syn303 antibody	BioLegend	824301	anti-mouse IF: 1:1000
Tau antibody	Synaptic Systems	314 002	anti-rabbit IF: 1:10,000
pser129-αS antibody	A gift from Fujiwara et al, reference 19		anti-rabbit WB: 1:5000
pser129-αS antibody	Abcam	ab51253	anti-rabbit IF: 1:1000
Mouse αS (D37A6) XP	Cell Signaling	4179	anti-rabbit IF 1:200
Alexa fluor 555-conjugated anti-rabbit antibody	Termo Fisher Scientific	A27039
Alexa fluor 488-conjugated anti-mouse antibody	Termo Fisher Scientific	A-11029
Microson XL-2000	Misonix		Sonicator
Ultra Bottles (Oakridge Bottles), PCB, 16x76mm, Assembly, Noryl Cap, Beckman-type	Science Service EU	S4484	Ultracentrifuge tubes
AXIO Observer Inverted Light Microscope	Zeiss	N/A
TCS SP2 laser scanning confocal microscope	Leica	N/A
Inverted epi-fluorescence microscope	Nikon	N/A
Triton x-100	Sigma-Aldrich	X100-500ML	Nonionic surfactant