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

Solji Choi; Bryan Killinger

May 21, 2024

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

DOI

dx.doi.org/10.17504/protocols.io.x54v92dq4l3e/v1

Solji Choi¹,
Bryan Killinger²

¹Rush University Medical Center;
²Rush University

Bryan Killinger

Rush University

DOI: dx.doi.org/10.17504/protocols.io.x54v92dq4l3e/v1

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

Funders Acknowledgement:

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:
AB
Tris-HCl, pH 7.450 mM
NaCl150 mM
Triton- X1000.5%

ReagentAlkaline Phosphatase, Calf Intestinal (20 u/μl)PromegaCatalog #M2825  

 CIAP buffer:
AB
NaCl100 mM
Tris-HCl50 mM
MgCl2, pH 7.910 mM
 Autoclave and store RT
 
 Blocking buffer:
AB
Dilution media100 mL
Normal serum3 mL
BSA2 g
Triton X1000.4 mL
Mix well so the Triton is completely dissolved
 
Borate buffer:
 
AB
Borate buffer, pH 8.50.05 M
DI H2O300 mL
Sodium tetraborate decahydrate5.72 g
Mix well to dissolve completely. Adjust to pH 8.5
 
Components:
AB
Borate buffer10 mL
H2O21 uL
TF5 uL
 
 Sodium Citrate Buffer, pH 6.0 (1L):   
AB
Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water2.94 g
Tween-200.5 mL
Mix well
 
PBS:
AB
Tris-HCl, pH 7.250 mM
NaCl158 mM

ReagentProteinase KThermo Fisher ScientificCatalog #EO0491 

Protocol materials

ReagentAlkaline Phosphatase, Calf Intestinal (20 u/μl)PromegaCatalog #M2825Materials, Step 5
 
ReagentProteinase KThermo Fisher ScientificCatalog #EO0491Materials, Step 25

Day 1

1d 1h 10m

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

Dilution media:
AB
Tris-HCl, pH 7.450 mM
NaCl150 mM
Triton- X1000.5%
 

Wash free-floating tissue for Duration00:10:00   in dilution media (DM). (1/3)

10m

Wash free-floating tissue for Duration00:10:00   in dilution media (DM). (2/3)

10m

Wash free-floating tissue for Duration00:10:00   in dilution media (DM). (3/3)

10m

Incubate the samples with 1% Triton X-100 in DM for Duration00:10:00  .

10m

Wash in DM for  Duration00:10:00  .

10m

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

CIAP buffer:
AB
NaCl100 mM
Tris-HCl50 mM
MgCl2, pH 7.910 mM
 Autoclave and store RT
 

Wash the tissues in CIAP buffer for Duration00:10:00  . (1/2)

10m

Wash the tissues in CIAP buffer for Duration00:10:00  . (2/2)

10m

Incubate the tissues with CIAP at a dilution of 1:333 for Duration24:00:00  at Temperature37 °C   on a shaker. 

ReagentAlkaline Phosphatase, Calf Intestinal (20 u/μl)PromegaCatalog #M2825 .
In 500uL CIAP buffer, add 1.5 μl CIAP (30 units). 

Day 2

8h 30m

Wash in DM (3 x 10 minutes).

Wash in DM for Duration00:10:00  . (1/3)

10m

Wash in DM for Duration00:10:00  . (2/3)

10m

Wash in DM for Duration00:10:00  . (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:
AB
Dilution media100 mL
Normal serum3 mL
BSA2 g
Triton X1000.4 mL
Mix well so the Triton is completely dissolved
 

Dilute primary antibody in blocking buffer. Incubate DurationOvernight   at Temperature4 °C  . 

Recombinant Anti-Alpha-synuclein (phospho S129) antibody (EP1536Y, ab51253), dilution factor: 1:50K
In Amount30 mL  blocking buffer, add Amount0.6 µL   PSER129 antibody.

Day 3

17h 20m

Wash in DM (3 x 10 minutes).

Wash in DM for Duration00:10:00  . (1/3)

10m

Wash in DM for Duration00:10:00  . (2/3)

10m

Wash in DM for Duration00:10:00  . (3/3)

10m

HRP-Secondary antibody incubation 1:1000 dilution (Duration01:00:00  ).  

Solvent is Amount100 mL   DM + Amount1 mL   normal serum + Amount1 g   BSA 

Wash in DM (2 x 10 minutes).

Wash in DM forDuration00:10:00  . (1/2)

10m

Wash in DM forDuration00:10:00  . (2/2)

10m

Wash in borate buffer for Duration00:10:00  . 

Borate buffer:
AB
Borate buffer, pH 8.50.05 M
DI H2O300 mL
Sodium tetraborate decahydrate5.72 g
Mix well to dissolve completely. Adjust to pH 8.5
 

10m

Incubate with tyramide fluorophore (TF) for Duration00:30:00   while blocking light.  After this step, always protect the tissues from light. 

Components:
AB
Borate buffer10 mL
H2O21 uL
TF5 uL
 

30m

Wash in DM (2 x 10 minutes).

Wash in DM Duration00:10:00   . (1/2)

10m

Wash in DM Duration00:10:00   . (2/2)

10m

View under the microscope to confirm successful staining.  

Heat water bath toTemperature80 °C  -Temperature85 °C   for  Duration01:30:00   before the primary antibody elution step. 

1h 30m

Place the dish containing sodium citrate buffer in the water bath and heat it for Duration00:10:00  . 

Sodium Citrate Buffer, pH 6.0 (1L):   
AB
Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water2.94 g
Tween-200.5 mL
Mix well
 

10m

Wash the tissues in sodium citrate buffer for Duration00:10:00   

10m

Incubate the tissues in the heated sodium citrate buffer forDuration00:30:00  . 

30m

Cool the dish containing tissues to TemperatureRoom temperature   (at least Duration00:20:00  ). 

20m

Wash in DM for 10 min x 2 times.

Wash in DM for Duration00:10:00   . (1/2)

10m

Wash in DM for Duration00:10:00   . (2/2)

10m

Mount the tissues on Superfrost Plus Microscope Slides (Fisherbrand), and completely dry it for at least Duration02:00:00  . 

Heat water bath to Temperature37 °C    for Duration00:30:00   before the PK digestion step.

30m

Place the dish containing PBS in the water bath and heat it for Duration00:10:00  . 

PBS: 
AB
Tris-HCl, pH 7.250 mM
NaCl158 mM
 

10m

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

ReagentProteinase KThermo Fisher ScientificCatalog #EO0491 
Amount30 mL   PBS, add Amount45 µL  PK.

Incubate the mounted tissues in the PK containing PBS for Duration00:30:00  . 

30m

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

Wash the slide in PBS for Duration00:05:00  . (1/2)

Wash the slide in PBS for Duration00:05:00  . (2/2)

Incubate the slide in 4% PFA for Duration00:30:00    at  TemperatureRoom temperature   on a shaker.

30m

Wash in DM (2 x 5 minutes).

Wash in DM for Duration00:05:00  . (1/2)

Wash in DM for Duration00:05:00  . (2/2)

Block the tissues on slide using Bloxall endogenous blocking solution (Vector Laboratories) for Duration00:10:00   at  TemperatureRoom temperature   on a shaker.

10m

Dilute primary antibody in blocking buffer. Incubate DurationOvernight   at Temperature4 °C  . 

Recombinant Anti-Alpha-synuclein antibody (EPR20535, ab212184), dilution factor: 1:20K
In Amount30 mL   blocking buffer, add Amount1.5 µL   aSyn antibody.

Day 4

3h 50m

Wash in DM (3 x 10 minutes).

Wash in DM for Duration00:10:00  . (1/3)

10m

Wash in DM for Duration00:10:00  . (2/3)

10m

Wash in DM for Duration00:10:00  . (3/3)

10m

HRP-Secondary antibody incubation 1:1000 dilution (Duration01:00:00  ).  

Solvent is Amount100 mL   DM + Amount1 mL   normal serum + Amount1 g   BSA 

Wash in DM (2 x 10 minutes).

Wash in DM for  Duration00:10:00  . (1/2)

10m

Wash in DM for  Duration00:10:00  . (2/2)

10m

Wash in borate buffer for Duration00:10:00  .

 Borate Buffer:
AB
Borate buffer, pH 8.50.05 M
DI H2O300 mL
Sodium tetraborate decahydrate5.72 g
 Mix well to dissolve completely. Adjust to pH 8.5
 

10m

Incubate with tyramide fluorophore (TF) for Duration00:30:00   while blocking light.  

30m

Wash in DM (2 x 10 minutes).

Wash in DM Duration00:10:00  . (1/2)

Wash in DM Duration00:10:00  . (2/2)

20m

 Components:

AB
Borate buffer10 mL
H2O21 uL
TF5 uL
 

View under the microscope to confirm successful staining.  

Wash in PBS (2 x 10 minutes).

Wash in PBS for Duration00:10:00  . (1/2)

10m

Wash in PBS for Duration00:10:00  . (2/2)

10m

Counterstain with DAPI for Duration00:20:00   at  TemperatureRoom temperature  . 

1:2000 dilution in ddH2O or PBS. 

20m

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

Wash the tissues in PBS for Duration00:10:00  . (1/2)

10m

Wash the tissues in PBS for Duration00:10:00  . (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 Temperature4 °C  .

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

	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

	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

	A	B
	Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water	2.94 g
	Tween-20	0.5 mL
	Mix well

	A	B
	Tris-HCl, pH 7.2	50 mM
	NaCl	158 mM

	A	B
	Tris-HCl, pH 7.2	50 mM
	NaCl	158 mM

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

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