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

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

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).

Heat water bath 1h 30m 0s before the antigen retrieval step.

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

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

a. Sodium Citrate Buffer, (1L): 6 (1L):

• 2.94g Sodium citrate-Trisodium salt (Dihydrate) in 1000mL DI water.
• Adjust pH to 6.0.
• 0.5mL Tween-20. Mix well.

Wash the tissues in sodium citrate buffer for 0h 5m 0s .

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

Cool down the tissues by placing container in an ice bucket for 0h 15m 0s.

Wash in DM for 10 minutes x 2 times.

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

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

Endogenous peroxidase inhibition and serum blocking step (1h 0m 0s incubation): 0.3% H₂O₂+0.1% Sodium Azide in blocking buffer.

a. Blocking buffer:

A	B
Dilution media	100 mL
Normal serum	3 mL
Bovine serum albumin	2 g
Triton x100 (Mix well so the Triton is completely dissolved)	0.4 mL

b. In 50 mL blocking buffer, add 0.5mL 30% H₂O₂ + 0.5mL10% Sodium Azide.

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

Wash (3 x 10 minutes) in dilution media.

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

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

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

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

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

Wash (2 x 10 minutes) in dilution media.

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

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

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

a. 0.05Molarity (M) Borate buffer 8.5

A	B
300mL	DI H2O
5.72 g	Sodium tetraborate decahydrate (P17, big bottle)

1. Mix well to dissolve completely.
2. Adjust to 8.5.

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

a. 10mL Borate buffer + 1µL H₂O₂ + 5µL TF.

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.

Heat water bath 1h 30m 0s before the antigen retrieval step.

a. Human samples: 90°C-95°C

b. Mouse samples: 80°C-85°C

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

a. Sodium Citrate Buffer, (1L): 6 (1L):

• 2.94g Sodium citrate-Trisodium salt (Dihydrate) in 1000mL DI water.
• Adjust pH to 6.0
• 0.5mLTween-20. Mix well.

Wash the tissues in sodium citrate buffer for 0h 5m 0s.

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

Cool down the tissues by placing container in an ice bucket for 0h 15m 0s.

Wash in DM for 10 minutes x 2 times.

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

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

Endogenous peroxidase inhibition and serum blocking step (0h 10m 0s incubation): 0.3% H₂O₂+0.1% Sodium Azide in blocking buffer.

a. Blocking buffer:

A	B
Dilution media	100 mL
Normal serum	3 mL
Bovine serum albumin	2 g
Triton x100 (Mix well so the Triton is completely dissolved)	0.4 mL

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

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

Wash (3 x 10 minutes) in dilution media.

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

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

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

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

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

Wash (2 x 10 minutes) in dilution media.

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

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

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

a. 0.05Molarity (M) Borate buffer 8.5

A	B
DI H2O	300mL
Sodium tetraborate decahydrate (P17, big bottle)	5.72 g

1. It takes a while to dissolve completely.
2. Adjust to 8.5.

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

a. 10mL Borate buffer + 1µL H₂O₂ + 5µL TF.

View under the microscope to confirm successful staining.

DAPI staining (0h 20m 0s)

a. 1:2000 dilution PBS. Block the light.

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.