May 21, 2024
Version 1
Multiplex Labeling with Tyramide Fluorophores for Detecting CIAP-Resistant PSER129 and Proteinase K-Resistant aSyn in situ (Killinger 2024) V.1
- 1Rush University Medical Center;
- 2Rush University
Protocol Citation: Solji Choi, Bryan Killinger 2024. Multiplex Labeling with Tyramide Fluorophores for Detecting CIAP-Resistant PSER129 and Proteinase K-Resistant aSyn in situ (Killinger 2024). protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92dq4l3e/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 15, 2024
Last Modified: May 21, 2024
Protocol Integer ID: 100194
Keywords: synuclein, m83 transgenic mice, intestinal alkaline phosphatase, resistant pser129, multiplex labeling with tyramide fluorophore, endogenous pser129, higher abundance of endogenous pser129, tyramide fluorophore, type mice, mice
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: ASAP-021030
NIH-NINDS
Grant ID: 1R01NS128467
Abstract
This protocol aims to examine the association of calf-intestinal alkaline phosphatase (CIAP)-resistant alpha-synuclein phosphorylated at serine 129 (PSER129) and proteinase K (PK)-resistant alpha-synuclein (aSyn) in the mouse brain, particularly in M83 transgenic mice treated with preformed fibrils. M83 lines exhibit a notably higher abundance of endogenous PSER129 compared to wild-type mice.
Materials
- Dilution media:
| A | B | |
| Tris-HCl, pH 7.4 | 50 mM | |
| NaCl | 150 mM | |
| Triton- X100 | 0.5% |
Alkaline Phosphatase, Calf Intestinal (20 u/μl)PromegaCatalog #M2825
- CIAP buffer:
| A | B | |
| NaCl | 100 mM | |
| Tris-HCl | 50 mM | |
| MgCl2, pH 7.9 | 10 mM | |
| Autoclave and store RT | ||
- 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 | ||
- 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 | ||
- Components:
| A | B | |
| Borate buffer | 10 mL | |
| H2O2 | 1 uL | |
| TF | 5 uL |
- 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 | ||
- PBS:
| A | B | |
| Tris-HCl, pH 7.2 | 50 mM | |
| NaCl | 158 mM |
Proteinase KThermo Fisher ScientificCatalog #EO0491
Protocol materials
Alkaline Phosphatase, Calf Intestinal (20 u/μl)PromegaCatalog #M2825
Proteinase KThermo Fisher ScientificCatalog #EO0491
Day 1
1d 1h 10m
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 00:10:00 in dilution media (DM). (1/3)
10m
Wash free-floating tissue for 00:10:00 in dilution media (DM). (2/3)
10m
Wash free-floating tissue for 00:10:00 in dilution media (DM). (3/3)
10m
Incubate the samples with 1% Triton X-100 in DM for 00:10:00 .
10m
Wash in DM for 00:10:00 .
10m
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 00:10:00 . (1/2)
10m
Wash the tissues in CIAP buffer for 00:10:00 . (2/2)
10m
Incubate the tissues with CIAP at a dilution of 1:333 for 24:00:00 at 37 °C on a shaker.
- Alkaline Phosphatase, Calf Intestinal (20 u/μl)PromegaCatalog #M2825 .
- In 500uL CIAP buffer, add 1.5 μl CIAP (30 units).
1d
Day 2
8h 30m
Wash in DM (3 x 10 minutes).
Wash in DM for 00:10:00 . (1/3)
10m
Wash in DM for 00:10:00 . (2/3)
10m
Wash in DM for 00:10:00 . (3/3)
10m
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 Overnight at 4 °C .
- Recombinant Anti-Alpha-synuclein (phospho S129) antibody (EP1536Y, ab51253), dilution factor: 1:50K
- In 30 mL blocking buffer, add 0.6 µL PSER129 antibody.
8h
Day 3
17h 20m
Wash in DM (3 x 10 minutes).
Wash in DM for 00:10:00 . (1/3)
10m
Wash in DM for 00:10:00 . (2/3)
10m
Wash in DM for 00:10:00 . (3/3)
10m
HRP-Secondary antibody incubation 1:1000 dilution (01:00:00 ).
- Solvent is 100 mL DM + 1 mL normal serum + 1 g BSA
1h
Wash in DM (2 x 10 minutes).
Wash in DM for00:10:00 . (1/2)
10m
Wash in DM for00:10:00 . (2/2)
10m
Wash in borate buffer for 00:10:00 .
- 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 | ||
10m
Incubate with tyramide fluorophore (TF) for 00:30:00 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 |
30m
Wash in DM (2 x 10 minutes).
Wash in DM 00:10:00 . (1/2)
10m
Wash in DM 00:10:00 . (2/2)
10m
View under the microscope to confirm successful staining.
Heat water bath to80 °C -85 °C for 01:30:00 before the primary antibody elution step.
1h 30m
Place the dish containing sodium citrate buffer in the water bath and heat it for 00:10:00 .
- 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 | ||
10m
Wash the tissues in sodium citrate buffer for 00:10:00
10m
Incubate the tissues in the heated sodium citrate buffer for00:30:00 .
30m
Cool the dish containing tissues to Room temperature (at least 00:20:00 ).
20m
Wash in DM for 10 min x 2 times.
Wash in DM for 00:10:00 . (1/2)
10m
Wash in DM for 00:10:00 . (2/2)
10m
Mount the tissues on Superfrost Plus Microscope Slides (Fisherbrand), and completely dry it for at least 02:00:00 .
2h
Heat water bath to 37 °C for 00:30:00 before the PK digestion step.
30m
Place the dish containing PBS in the water bath and heat it for 00:10:00 .
- PBS:
| A | B | |
| Tris-HCl, pH 7.2 | 50 mM | |
| NaCl | 158 mM |
10m
Add the PK to the PBS at a dilution of 1:666 and mix well.
- Proteinase KThermo Fisher ScientificCatalog #EO0491
- 30 mL PBS, add 45 µL PK.
Incubate the mounted tissues in the PK containing PBS for 00:30:00 .
30m
Wash the slide in PBS (2 x 5 minutes).
Wash the slide in PBS for 00:05:00 . (1/2)
5m
Wash the slide in PBS for 00:05:00 . (2/2)
5m
Incubate the slide in 4% PFA for 00:30:00 at Room temperature on a shaker.
30m
Wash in DM (2 x 5 minutes).
Wash in DM for 00:05:00 . (1/2)
5m
Wash in DM for 00:05:00 . (2/2)
5m
Block the tissues on slide using Bloxall endogenous blocking solution (Vector Laboratories) for 00:10:00 at Room temperature on a shaker.
10m
Dilute primary antibody in blocking buffer. Incubate Overnight at 4 °C .
- Recombinant Anti-Alpha-synuclein antibody (EPR20535, ab212184), dilution factor: 1:20K
- In 30 mL blocking buffer, add 1.5 µL aSyn antibody.
8h
Day 4
3h 50m
Wash in DM (3 x 10 minutes).
Wash in DM for 00:10:00 . (1/3)
10m
Wash in DM for 00:10:00 . (2/3)
10m
Wash in DM for 00:10:00 . (3/3)
10m
HRP-Secondary antibody incubation 1:1000 dilution (01:00:00 ).
- Solvent is 100 mL DM + 1 mL normal serum + 1 g BSA
1h
Wash in DM (2 x 10 minutes).
Wash in DM for 00:10:00 . (1/2)
10m
Wash in DM for 00:10:00 . (2/2)
10m
Wash in borate buffer for 00:10:00 .
- 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 | ||
10m
Incubate with tyramide fluorophore (TF) for 00:30:00 while blocking light.
30m
Wash in DM (2 x 10 minutes).
- Wash in DM 00:10:00 . (1/2)
- Wash in DM 00:10:00 . (2/2)
20m
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 00:10:00 . (1/2)
10m
Wash in PBS for 00:10:00 . (2/2)
10m
Counterstain with DAPI for 00:20:00 at Room temperature .
- 1:2000 dilution in ddH2O or PBS.
20m
Wash the tissues in PBS (2 x 10 minutes).
Wash the tissues in PBS for 00:10:00 . (1/2)
10m
Wash the tissues in PBS for 00:10:00 . (2/2)
10m
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 .