Apr 02, 2024

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

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvme7zng3p/v1
  • Bryan Killinger1,
  • Tyler Tittle1,
  • Solji Choi2
  • 1Rush University;
  • 2Rush University Medical Center
Bryan Killinger
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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...
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Materials
Sodium Citrate Buffer,Ph6 (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)
Concentration0.05 Molarity (M) Borate buffer Ph8.5

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

10m
Wash
Wash free-floating tissue for Duration00:10:00 in dilution media (DM) (2/3).
10m
Wash
Wash free-floating tissue for Duration00:10:00 in dilution media (DM) (3/3).
10m
Wash
Heat water bath Duration01:30:00 before the antigen retrieval step.

  1. Human samples: Temperature90 °C -Temperature95 °C
  2. Mouse samples: Temperature80 °C -Temperature85 °C

1h 30m
Temperature
Place the dish containing sodium citrate buffer in the water bath and heat it for Duration00:10:00 .

a. Sodium Citrate Buffer,Ph6 (1L):
  • Amount2.94 g Sodium citrate-Trisodium salt (Dihydrate) in Amount1000 mL DI water.
  • Adjust pH to 6.0.
  • Amount0.5 mL Tween-20. Mix well.
10m
Mix
Wash the tissues in sodium citrate buffer for Duration00:05:00 .
5m
Wash
Incubate the tissues in the heated sodium citrate buffer for Duration00:30:00 .
30m
Incubation
Cool down the tissues by placing container in an ice bucket for Duration00:15:00 .
15m
Wash in DM for 10 minutes x 2 times.

Wash
Wash in DM for Duration00:10:00 (1/2).
10m
Wash
Wash in DM for Duration00:10:00 (2/2).
10m
Wash
Endogenous peroxidase inhibition and serum blocking step (Duration01:00:00 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 Amount0.5 mL 30% H2O2 + Amount0.5 mL 10% Sodium Azide.

1h
Incubation
Pipetting
Dilute primary antibody in blocking buffer. Incubate DurationOvernight at Temperature4 °C .

10m
Incubation
Overnight
Temperature
Day 2:
2w 0d 2h
Wash (3 x 10 minutes) in dilution media.
Wash
Wash for Duration00:10:00 in dilution media (1/3).
10m
Wash
Wash for Duration00:10:00 in dilution media (2/3).
10m
Wash
Wash for Duration00:10:00 in dilution media (3/3).
10m
Wash
HRP-Secondary antibody incubation 1:1000 dilution (Duration01:00:00 ).

  • Solvent is Amount100 mL DM/Amount1 mL normal serum/Amount1 g BSA.

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

Wash
Wash for Duration00:10:00 in dilution media (1/2).
10m
Wash
Wash for Duration00:10:00 in dilution media (2/2).
10m
Wash
Wash in borate buffer for Duration00:10:00 .

a. Concentration0.05 Molarity (M) Borate buffer Ph8.5

AB
300mL DI H2O
5.72 gSodium tetraborate decahydrate (P17, big bottle)
  1. Mix well to dissolve completely.
  2. Adjust to Ph8.5 .
10m
Pipetting
Wash
Mix
Incubate with tyramide fluorophore (TF) for Duration00:30:00 while blocking light.

a. Amount10 mL Borate buffer + Amount1 µL H2O2 + Amount5 µL TF.
30m
Incubation
View under the microscope to confirm successful staining.
Store in PBS and leave at Temperature4 °C . It can be stored for up to 2 weeks. Otherwise, proceed with the antigen retrieval step.
Temperature
Day 3:
2h 50m
Heat water bath Duration01:30:00 before the antigen retrieval step.

a. Human samples: Temperature90 °C -Temperature95 °C
b. Mouse samples: Temperature80 °C -Temperature85 °C

1h 30m
Temperature
Place the dish containing sodium citrate buffer in the water bath and heat it for Duration00:10:00 .

a. Sodium Citrate Buffer,Ph6 (1L):
  • Amount2.94 g Sodium citrate-Trisodium salt (Dihydrate) in Amount1000 mL DI water.
  • Adjust pH to 6.0
  • Amount0.5 mL Tween-20. Mix well.



10m
Wash the tissues in sodium citrate buffer for Duration00:05:00 .
5m
Wash
Incubate the tissues in the heated sodium citrate buffer for Duration00:30:00 .
30m
Incubation
Cool down the tissues by placing container in an ice bucket for Duration00:15:00 .
15m
Wash in DM for 10 minutes x 2 times.
Wash
Wash in DM for Duration00:10:00 (1/2).
10m
Wash
Wash in DM for Duration00:10:00 (2/2).
10m
Wash
Endogenous peroxidase inhibition and serum blocking step (Duration00:10:00 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 Amount0.5 mL 30% H2O2 + Amount0.5 mL 10% Sodium Azide.

10m
Incubation
Dilute primary antibody in blocking buffer. Incubate DurationOvernight at Temperature4 °C .
10m
Incubation
Overnight
Temperature
Day 4:
2h 20m
Wash (3 x 10 minutes) in dilution media.
Wash
Wash for Duration00:10:00 in dilution media (1/3).
10m
Wash
Wash for Duration00:10:00 in dilution media (2/3).
10m
Wash
Wash for Duration00:10:00 in dilution media (3/3).
10m
Wash
HRP-Secondary antibody incubation 1:1000 dilution (Duration01:00:00 ).

a. Solvent is Amount100 mL DM/1 mL normal serum/1g BSA.

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

Wash for Duration00:10:00 in dilution media (1/2).
10m
Wash for Duration00:10:00 in dilution media (2/2).
10m
Wash in borate buffer for Duration00:10:00 .

a. Concentration0.05 Molarity (M) Borate buffer Ph8.5
AB
DI H2O300mL 
Sodium tetraborate decahydrate (P17, big bottle)5.72 g
  1. It takes a while to dissolve completely.
  2. Adjust to Ph8.5 .
10m
Pipetting
Wash
Incubate with tyramide fluorophore (TF) on a shaker for Duration00:30:00 while blocking light.

a. Amount10 mL Borate buffer + Amount1 µL H2O2 + Amount5 µL TF.
30m
Incubation
Pipetting
View under the microscope to confirm successful staining.
Imaging
DAPI staining (Duration00:20:00 )

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 Temperature4 °C .
Imaging
Temperature