Apr 02, 2024

Multiplex Labeling with Tyramide Fluorophores (Free-Floating Tissues)-Killinger Lab 2024

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvme7zng3p/v1
  • 1Rush University;
  • 2Rush University Medical Center
Bryan Killinger
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.yxmvme7zng3p/v1
Protocol Citation: Bryan Killinger, Tyler Tittle, Solji Choi 2024. Multiplex Labeling with Tyramide Fluorophores (Free-Floating Tissues)-Killinger Lab 2024. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvme7zng3p/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: March 18, 2024
Last Modified: April 02, 2024
Protocol  Integer ID: 97660
Keywords: multiplex labeling with tyramide fluorophore, tyramide fluorophores in the killinger lab, using tyramide fluorophore, multiplex labeling, tyramide fluorophore, floating tissue, killinger lab, tissue, lab 2024 this protocol detail, lab
Funders Acknowledgements:
NIH-NINDS
Grant ID: 1R01NS128467
Michael J Fox Foundation
Grant ID: ASAP-024442
Abstract
This protocol details the multiplex labeling of free-floating tissues using tyramide fluorophores in the killinger lab (2024).
Attachments
Icon for 2024 Dual Labeling Using Tyramide Fluorophores.docx
2024 Dual Labeling U...
18KB
Materials
Sodium Citrate Buffer,6 (1L):
AB
2.94 g Sodium citrate-Trisodium salt(Dihydrate)in 1000 mL DI water.
6.0 pHAdjust pH to
0.5 mL Tween-20 (Mix well)
Blocking buffer:

AB
100 mL Dilution media
3 mLNormal serum
2 gBovine serum albumin
0.4 mLTriton x100 (Mix well so the Triton is completely dissolved)
0.05 Mass Percent Borate buffer 8.5

AB
300mL DI H2O
5.72 gSodium tetraborate decahydrate (P17, big bottle)
Day 1:
1h 50m
Wash free-floating tissue (3 x 10 minutes) in dilution media (DM).

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
Heat water bath 01:30:00 before the antigen retrieval step.

  1. Human samples: 90 °C -95 °C
  2. Mouse samples: 80 °C -85 °C

1h 30m
Place the dish containing sodium citrate buffer in the water bath and heat it for 00:10:00 .

a. Sodium Citrate Buffer,6 (1L):
  • 2.94 g Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water.
  • Adjust pH to 6.0.
  • 0.5 mL Tween-20. Mix well.
10m
Wash the tissues in sodium citrate buffer for 00:05:00 .
5m
Incubate the tissues in the heated sodium citrate buffer for 00:30:00 .
30m
Cool down the tissues by placing container in an ice bucket for 00:15:00 .
15m
Wash in DM for 10 minutes x 2 times.

Wash in DM for 00:10:00 (1/2).
10m
Wash in DM for 00:10:00 (2/2).
10m
Endogenous peroxidase inhibition and serum blocking step (01:00:00 incubation): 0.3% H2O2+0.1% Sodium Azide in blocking buffer.

a. Blocking buffer:
AB
Dilution media100 mL 
Normal serum3 mL
Bovine serum albumin2 g
Triton x100 (Mix well so the Triton is completely dissolved)0.4 mL


b. In 50 mL blocking buffer, add 0.5 mL 30% H2O2 + 0.5 mL 10% Sodium Azide.

1h
Dilute primary antibody in blocking buffer. Incubate Overnight at 4 °C .

10m
Day 2:
2w 0d 2h
Wash (3 x 10 minutes) in dilution media.
Wash for 00:10:00 in dilution media (1/3).
10m
Wash for 00:10:00 in dilution media (2/3).
10m
Wash for 00:10:00 in dilution media (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 (2 x 10 minutes) in dilution media.

Wash for 00:10:00 in dilution media (1/2).
10m
Wash for 00:10:00 in dilution media (2/2).
10m
Wash in borate buffer for 00:10:00 .

a. 0.05 Mass Percent Borate buffer 8.5

AB
300mL DI H2O
5.72 gSodium tetraborate decahydrate (P17, big bottle)
  1. Mix well to dissolve completely.
  2. Adjust to 8.5 .
10m
Incubate with tyramide fluorophore (TF) for 00:30:00 while blocking light.

a. 10 mL Borate buffer + 1 µL H2O2 + 5 µL TF.
30m
View under the microscope to confirm successful staining.
Store in PBS and leave at 4 °C . It can be stored for up to 2 weeks. Otherwise, proceed with the antigen retrieval step.
Day 3:
2h 50m
Heat water bath 01:30:00 before the antigen retrieval step.

a. Human samples: 90 °C -95 °C
b. Mouse samples: 80 °C -85 °C

1h 30m
Place the dish containing sodium citrate buffer in the water bath and heat it for 00:10:00 .

a. Sodium Citrate Buffer,6 (1L):
  • 2.94 g Sodium citrate-Trisodium salt (Dihydrate) in 1000 mL DI water.
  • Adjust pH to 6.0
  • 0.5 mL Tween-20. Mix well.



10m
Wash the tissues in sodium citrate buffer for 00:05:00 .
5m
Incubate the tissues in the heated sodium citrate buffer for 00:30:00 .
30m
Cool down the tissues by placing container in an ice bucket for 00:15:00 .
15m
Wash in DM for 10 minutes x 2 times.
Wash in DM for 00:10:00 (1/2).
10m
Wash in DM for 00:10:00 (2/2).
10m
Endogenous peroxidase inhibition and serum blocking step (00:10:00 incubation): 0.3% H2O2+0.1% Sodium Azide in blocking buffer.

a. Blocking buffer:
AB
Dilution media100 mL 
Normal serum3 mL
Bovine serum albumin2 g
Triton x100 (Mix well so the Triton is completely dissolved)0.4 mL
b. In 50 mL blocking buffer, add 0.5 mL 30% H2O2 + 0.5 mL 10% Sodium Azide.

10m
Dilute primary antibody in blocking buffer. Incubate Overnight at 4 °C .
10m
Day 4:
2h 20m
Wash (3 x 10 minutes) in dilution media.
Wash for 00:10:00 in dilution media (1/3).
10m
Wash for 00:10:00 in dilution media (2/3).
10m
Wash for 00:10:00 in dilution media (3/3).
10m
HRP-Secondary antibody incubation 1:1000 dilution (01:00:00 ).

a. Solvent is 100 mL DM/1 mL normal serum/1g BSA.

1h
Wash (2 x 10 minutes) in dilution media.

Wash for 00:10:00 in dilution media (1/2).
10m
Wash for 00:10:00 in dilution media (2/2).
10m
Wash in borate buffer for 00:10:00 .

a. 0.05 Mass Percent Borate buffer 8.5
AB
DI H2O300mL 
Sodium tetraborate decahydrate (P17, big bottle)5.72 g
  1. It takes a while to dissolve completely.
  2. Adjust to 8.5 .
10m
Incubate with tyramide fluorophore (TF) on a shaker for 00:30:00 while blocking light.

a. 10 mL Borate buffer + 1 µL H2O2 + 5 µL TF.
30m
View under the microscope to confirm successful staining.
DAPI staining (00:20:00 )

a. 1:2000 dilution PBS. Block the light.
20m
Mount the tissues on a slide, cover the slide with Fluoroshield, and coverslip. Seal with nail polish on all sides of the coverslip.
When the nail polish is completely dried, view under the microscope. Always protect the slides from light. Slides can be stored at 4 °C .