Multiplex Labeling with Tyramide Fluorophores for Detecting CIAP-Resistant PSER129 and Proteinase K-Resistant aSyn in situ (Killinger 2024)

Wash free-floating tissue (3 x 10 minutes) in dilution media (DM).

Dilution media:

A	B
Tris-HCl, pH 7.4	50 mM
NaCl	150 mM
Triton- X100	0.5%

Wash free-floating tissue for 0h 10m 0s in dilution media (DM). (1/3)

Wash free-floating tissue for 0h 10m 0s in dilution media (DM). (2/3)

Wash free-floating tissue for 0h 10m 0s in dilution media (DM). (3/3)

Incubate the samples with 1% Triton X-100 in DM for 0h 10m 0s.

Wash in DM for 0h 10m 0s.

Wash the tissues in CIAP buffer (2x10 minutes).

CIAP buffer:

A	B
NaCl	100 mM
Tris-HCl	50 mM
MgCl2, pH 7.9	10 mM
Autoclave and store RT

Wash the tissues in CIAP buffer for 0h 10m 0s. (1/2)

Wash the tissues in CIAP buffer for 0h 10m 0s. (2/2)

Incubate the tissues with CIAP at a dilution of 1:333 for 24h 0m 0sat 37°C on a shaker.

• .
• In 500uL CIAP buffer, add 1.5 μl CIAP (30 units).

Wash in DM (3 x 10 minutes).

Wash in DM for 0h 10m 0s. (1/3)

Wash in DM for 0h 10m 0s. (2/3)

Wash in DM for 0h 10m 0s. (3/3)

Endogenous peroxidase inhibition and serum blocking step (1-hour incubation):

• 0.3% H₂O₂+0.1% Sodium Azide in 50 mL blocking buffer.

• Blocking buffer: | A | B | | --- | --- | | Dilution media | 100 mL | | Normal serum | 3 mL | | BSA | 2 g | | Triton X100 | 0.4 mL | | Mix well so the Triton is completely dissolved | |

Dilute primary antibody in blocking buffer. Incubate 0h 10m 0s at 4°C.

• Recombinant Anti-Alpha-synuclein (phospho S129) antibody (EP1536Y, ab51253), dilution factor: 1:50K
• In 30mLblocking buffer, add 0.6µL PSER129 antibody.

Wash in DM (3 x 10 minutes).

Wash in DM for 0h 10m 0s. (1/3)

Wash in DM for 0h 10m 0s. (2/3)

Wash in DM for 0h 10m 0s. (3/3)

HRP-Secondary antibody incubation 1:1000 dilution (1h 0m 0s).

• Solvent is 100mL DM + 1mL normal serum + 1g BSA

Wash in DM (2 x 10 minutes).

Wash in DM for0h 10m 0s. (1/2)

Wash in DM for0h 10m 0s. (2/2)

Wash in borate buffer for 0h 10m 0s.

• Borate buffer: | A | B | | --- | --- | | Borate buffer, pH 8.5 | 0.05 M | | DI H2O | 300 mL | | Sodium tetraborate decahydrate | 5.72 g | | Mix well to dissolve completely. Adjust to pH 8.5 | |

Incubate with tyramide fluorophore (TF) for 0h 30m 0s while blocking light. After this step, always protect the tissues from light.

• Components: | A | B | | --- | --- | | Borate buffer | 10 mL | | H2O2 | 1 uL | | TF | 5 uL |

Wash in DM (2 x 10 minutes).

Wash in DM 0h 10m 0s . (1/2)

Wash in DM 0h 10m 0s . (2/2)

View under the microscope to confirm successful staining.

Heat water bath to80°C-85°C for 1h 30m 0s before the primary antibody elution step.

Place the dish containing sodium citrate buffer in the water bath and heat it for 0h 10m 0s.

• Sodium Citrate Buffer, pH 6.0 (1L): | A | B | | --- | --- | | Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water | 2.94 g | | Tween-20 | 0.5 mL | | Mix well | |

Wash the tissues in sodium citrate buffer for 0h 10m 0s

Incubate the tissues in the heated sodium citrate buffer for0h 30m 0s.

Cool the dish containing tissues to Room temperature (at least 0h 20m 0s).

Wash in DM for 10 min x 2 times.

Wash in DM for 0h 10m 0s . (1/2)

Wash in DM for 0h 10m 0s . (2/2)

Mount the tissues on Superfrost Plus Microscope Slides (Fisherbrand), and completely dry it for at least 2h 0m 0s.

Heat water bath to 37°C for 0h 30m 0s before the PK digestion step.

Place the dish containing PBS in the water bath and heat it for 0h 10m 0s.

• PBS: | A | B | | --- | --- | | Tris-HCl, pH 7.2 | 50 mM | | NaCl | 158 mM |

Add the PK to the PBS at a dilution of 1:666 and mix well.

• 30mL PBS, add 45µLPK.

Incubate the mounted tissues in the PK containing PBS for 0h 30m 0s.

Wash the slide in PBS (2 x 5 minutes).

Wash the slide in PBS for 0h 5m 0s. (1/2)

Wash the slide in PBS for 0h 5m 0s. (2/2)

Incubate the slide in 4% PFA for 0h 30m 0s at Room temperature on a shaker.

Wash in DM (2 x 5 minutes).

Wash in DM for 0h 5m 0s. (1/2)

Wash in DM for 0h 5m 0s. (2/2)

Block the tissues on slide using Bloxall endogenous blocking solution (Vector Laboratories) for 0h 10m 0s at Room temperature on a shaker.

Dilute primary antibody in blocking buffer. Incubate 0h 10m 0s at 4°C.

• Recombinant Anti-Alpha-synuclein antibody (EPR20535, ab212184), dilution factor: 1:20K
• In 30mL blocking buffer, add 1.5µL aSyn antibody.

Wash in DM (3 x 10 minutes).

Wash in DM for 0h 10m 0s. (1/3)

Wash in DM for 0h 10m 0s. (2/3)

Wash in DM for 0h 10m 0s. (3/3)

HRP-Secondary antibody incubation 1:1000 dilution (1h 0m 0s).

• Solvent is 100mL DM + 1mL normal serum + 1g BSA

Wash in DM (2 x 10 minutes).

Wash in DM for 0h 10m 0s. (1/2)

Wash in DM for 0h 10m 0s. (2/2)

Wash in borate buffer for 0h 10m 0s.

• Borate Buffer: | A | B | | --- | --- | | Borate buffer, pH 8.5 | 0.05 M | | DI H2O | 300 mL | | Sodium tetraborate decahydrate | 5.72 g | |  Mix well to dissolve completely. Adjust to pH 8.5 | |

Incubate with tyramide fluorophore (TF) for 0h 30m 0s while blocking light.

Wash in DM (2 x 10 minutes).

• Wash in DM 0h 10m 0s. (1/2)

• Wash in DM 0h 10m 0s. (2/2)

Components:

A	B
Borate buffer	10 mL
H2O2	1 uL
TF	5 uL

View under the microscope to confirm successful staining.

Wash in PBS (2 x 10 minutes).

Wash in PBS for 0h 10m 0s. (1/2)

Wash in PBS for 0h 10m 0s. (2/2)

Counterstain with DAPI for 0h 20m 0s at Room temperature.

• 1:2000 dilution in ddH₂O or PBS.

Wash the tissues in PBS (2 x 10 minutes).

Wash the tissues in PBS for 0h 10m 0s. (1/2)

Wash the tissues in PBS for 0h 10m 0s. (2/2)

Cover the slide with Fluoroshield, and coverslip. Seal with nail polish on all sides of the coverslip. Always protect the slides from light. Slides can be stored at 4°C.