An Immunofluorescence Assay to Detect Alpha-Synuclein Aggregates in a Skin Biopsy Section

Overview

This video demonstrates an immunofluorescence assay to detect neurodegenerative disease-specific alpha-synuclein aggregates on skin biopsy sections. Free-floating immunostaining is employed to fluorescently label the carboxyl-terminal hydrolase and the alpha-synuclein aggregates in the dermal neurons. Under a confocal microscope, cells with fluorescence from both antibodies indicate the presence of alpha-synuclein aggregates in the dermal neurons.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines,

1. Skin Biopsy Collection

1. Let a qualified physician perform the skin biopsy in an appropriate clinical setting.
2. Choose the area to perform the skin biopsy and clean it with an alcohol swab.
3. Prepare the anesthetic solution with 1 cc of lidocaine 2%.
4. With the needle parallel to the area, inject local anesthesia subcutaneously.
5. After checking the effect of anesthesia, take the 3 mm disposable punch and apply rotational and delicate downward pressure to rotate it down through the epidermis and dermis, until the subcutaneous fat is reached.
6. Withdraw the punch.
7. Use disposable forceps to gently pull the skin plug up.
8. Use scissors to cut out the base of the specimen at the level of the fatty tissue.
9. Place the specimen in a tube containing 10 mL of periodate-lysine-paraformaldehyde (PLP) fixative solution.
10. Disinfect the area and cover it with an adhesive bandage.

2. Tissue Fixation and Storage

1. Make fresh PLP fixative solution (see Table 1).
2. Immediately after the collection of the skin biopsy, submerge it in a tube containing 10 mL of PLP fixative solution and incubate it overnight (O/N) at 4 °C.
3. The day after, under the fume hood, remove the PLP fixative gently and in the same tube, wash the biopsy 3 times for 5 min with 5 mL of 0.1 M Sorensen's solution (Table 1).
4. Discard the Sorensen's solution and incubate the biopsy with 5 mL of cryo-protectant solution O/N at 4 °C.
5. Store the biopsy at 4 °C if the cut with a cryotome is performed within 1 week; store the biopsy at -20 °C if the cut is performed within 3 months; embed the biopsy in a cryomold (steps 3.2-3.4) and store it at -80 °C to conserve it for a longer period of time.

3. Tissue Cut with a Cryotome

1. Set the cryotome at -20 °C.
2. Take a cryomold and fill it up with the cryo-embedding medium. Avoid creating bubbles.
3. Using tweezers immerse the biopsy into the cryo-embedding medium with the longitudinal axis (epidermis - dermis) parallel to the bottom of the cryomold.
4. Snap freeze the sample with liquid nitrogen to obtain a solid cube of cryo-embedding medium containing the biopsy in the right orientation.
5. Put the sample in the cryostat and wait for 30 min to allow the biopsy to acclimatize.
6. Fix the sample on the cryostat and cut 50 µm sections.
7. With the help of a little brush, transfer the cryo-sections in a 96 well plate containing 200 µL of antifreeze solution in each well. One section per well.
8. Store at -20 °C.
   NOTE: If the analyze the entire biopsy is not analyzed, cut it only partially. In this case, the sections must be stored at -20 °C and the remaining part of the biopsy at -80 °C to conserve it for a longer period of time.

4. Immunofluorescence Staining

1. Fill a 96 well plate, with 100 µL of washing solution.
2. Transfer the sections to be analyzed from the storage plate to the new one containing the washing solution. 1 section per each well (4 sections per anatomical site, per patient: usually a total of 12 sections per patient).
3. Leave the section in the washing solution for 10 min at room temperature (RT). Transfer the section into another well containing the same solution and repeat the wash.
4. Move the sections into new wells containing 100 µL of Blocking solution and incubate for at least 90 min and for a maximum of 4 h at RT.
5. Dilute the primary antibodies anti-PGP9.5 (Rabbit polyclonal, 1:1000) and anti-5G4 (Mouse monoclonal, 1:400) in the working solution.
6. Transfer the sections into new wells containing 100 µL of the working solution of the primary antibodies and incubate them O/N at RT.
7. As step 4.3, wash the sections 2 times for at least 10 min at RT, with 100 µL of washing solution.
8. Dilute the secondary antibodies (conjugate with different fluorophores) Goat anti-Rabbit to detect PGP9.5 (1:700) and a Goat anti-Mouse to detect 5G4 (1:700) in the working solution.
9. Transfer the sections into new wells containing 100 µL of the working solution of the secondary antibodies for 90 min at RT. From this point, cover the 96 well plate with aluminum foil to avoid the bleaching of fluorophore conjugated with secondary antibody/ies.
10. As step 4.3, wash the sections 2 times for at least 10 min, with 100 µL of the washing solution.
11. Transfer the sections into new wells containing 100 µL of 4′,6-diamidino-2-phenylindole (DAPI, diluted 1:5,000 in phosphate-buffered saline [PBS] 1x) for 5 min at RT.
12. As step 4.3, wash the sections 2 times for at least 10 min, with 100 µL of the washing solution.
13. Mount the sections on a slide in the correct position avoiding misfolding.
14. Add a few drops of the mounting medium on the slide and cover with a coverslip.
15. Let slides dry O/N before using the confocal/fluorescence microscope.
16. Store the slides in an appropriate box at 4 °C avoiding the light exposure. If accurately stored, the signal will be visible for about 6 months.
    NOTE: The transfer of a section from the 96 well plate containing the newly cut slices to the 96 well plate containing the washing solution (step 4.1) is performed using a small brush that helps to pick up the biopsy. After the transfer of the section into the first well, every time the slice needs to be incubated with a different solution, move it from well to well with the help of the brush.

5. Immunofluorescence Imaging

1. View sections under an inverted fluorescence microscope or a confocal microscope (20X, 40x or higher magnification, use successive frames of 2 µm increments on a Z-stack plan for best results).
2. Acquire images by a microscope connected camera
3. Use an adequate imaging software (i.e. ImageJ) to analyze positive signals in sections in terms of spatial distribution and intensity of the signal. To do this perform the steps mentioned below.
   1. Open the file with ImageJ software.
   2. Click on Image > color > split channels in order to analyze each color channel.
   3. Click Image > stack > Z project to obtain a merge of multiple section acquisitions.
   4. Click Image > color > merge channels to obtain a merge of different color channels of the same acquisition.

Table 1: Required solutions. List of required solutions for the protocol and brief description of how to prepare it.

Antifreeze solution (store at 4 °C for up to 6 months)	30% Glycerol 30% Ethylene glycol 30% dH2O 10% 2x Phosphate buffer
Blocking solution (prepare at the moment)	4% Normal Goat Serum 1% Triton X-100 in Washing solution
Cryo-protectant (store at 4 °C for up to 6 months)	20% Glycerol 80% Sorensen's solution
Disodium hydrogen phosphate solution (store at RT up to 6 months)	0.45M Disodium hydrogen phosphate (Na2HPO4) in dH2O Filter in a sterile bottle
Lysine solution (store at 4 °C for up to 3 weeks)	50% of 0.3M L-Lysine monohydrochloride solution 50% of 0.1M Sorensen's solution pH 7.6 pH 7.4, filter in a sterile bottle
Paraformaldehyde (PFA) 8% (prepare under fume hood and store at 4 °C for up to 1 month)	2.6M PFA in dH2O (to 55°C_do not exceed 60 °C to avoid formic acid formation) filter in a sterile bottle
Phosphate buffer 2x (store at 4 °C for up to 6 months)	6% Monosodium phosphate (NaH2PO4) solution 45% Disodium phosphate (Na2HPO4) solution in dH2O
PLP fixative solution (prepare at the moment, under fume hood )	25% Paraformaldehyde 8% 0.01M Sodium (meta)periodate 75% Lysine solution
Sodium Dihydrogen Phosphate Monohydrate (store at RT up to 6 months)	0.52M Sodium Dihydrogen Phosphate Monohydrate (NaH2PO4*H2O) in dH2O Filter in a sterile bottle.
Sorensen's solution (store at 4 °C for up to 1 month)	2.5% Monosodium phosphate solution 18.7% Disodium phosphate solution pH 7.6, in dH2O
Washing solution (prepare at the moment)	0.25M Trizma base 0.26M NaCl pH 7.6, in dH2O
Working solution (prepare at the moment)	50% Blocking solution 50% Washing solution

Materials

Name	Company	Catalog Number	Comments
5G4 (anti human α-Synuclein 5G4)	Analytik Jena Roboscreen	847-0102004001	Mouse monoclonal
AlexaFluor 488 Goat anti Rabbit IgG	Invitrogen	1971418	2mg/ml
AlexaFluor 594 Goat anti Mouse IgG	Invitrogen	1922849	2mg/ml
Disodium hydrogen phosphate solution	Merk Millipore	106586
Ethylene Glycol	Sigma-Aldrich	324558
Glycerol	Sigma-Aldrich	G7757
L-Lysine monohydrochloride	Sigma-Aldrich	L5626
Paraformaldehyde	Aldrich Chemistry	441244
PGP9.5	Abcam	ab15503	Rabbit polyclonal
Sodium Chloride	Sigma	S3014
Sodium Dihydrogen Phosphate Monohydrate	Merck Millipore	106346
Sodium (meta)periodate	Sigma-Aldrich	S1878
Trizma Base	Sigma	T1503
Tryton X-100	Sigma-Aldrich	X100
Vectashield	Vector Laboratories	H-1000	Mounting medium