TH DAB staining

Pranay Srivastava, Waijiao Cai, Xiqun Chen

Published: 2022-07-16 DOI: 10.17504/protocols.io.j8nlk4yw1g5r/v1

TH DAB staining

TBST

ASAPCRN

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Abstract

This protocol details the stating procedure for TH DAB staining.

Attachments

ds8ybjtc7.pdf

Steps

TH DAB staining

Wash a full set of sections (6-8 sections) in a well in PBS, 5 min × 3.

1.1.

Wash a full set of sections (6-8 sections) in a well in PBS, 0h 5m 0s (wash 1/3) (if stored in cryoprotective solution).

1.2.

Wash a full set of sections (6-8 sections) in a well in PBS, 0h 5m 0s (wash 2/3) (if stored in cryoprotective solution).

1.3.

Wash a full set of sections (6-8 sections) in a well in PBS, 0h 5m 0s (wash 3/3) (if stored in cryoprotective solution).

Incubate sections in 1% H₂O₂ (1mL 30% H₂O₂ in 29mL TBST), 0h 30m 0s at Room temperature.

Wash sections in TBST 5 min × 3.

3.1.

Wash sections in TBST 0h 5m 0s (wash 1/3).

3.2.

Wash sections in TBST 0h 5m 0s (wash 2/3).

3.3.

Wash sections in TBST 0h 5m 0s (wash 3/3).

Block sections in 5% NGS/TBST, 1h 0m 0s at Room temperature.

Incubate sections in rabbit anti-TH (#SA-497), 1:3000 in TBST, 4°C 1h 0m 0s.

Wash sections in TBST, 10 min × 3.

6.1.

Wash sections in TBST, 0h 10m 0s (wash 1/3).

6.2.

Wash sections in TBST, 0h 10m 0s (wash 2/3).

6.3.

Wash sections in TBST, 0h 10m 0s (wash 3/3).

Incubate sections in anti-rabbit biotinylated (#OS03B), 1:3000, 1h 0m 0s at 4Room temperature.

Prepare ABC (Avidin-Biotin-Complex, Vectastain ABC Kit, # PK-6100): 1 drop A and 1 drop B in 2.5mL TBST, at least 30 min before use.

Wash sections in TBST, 10 min × 3.

9.1.

Wash sections in TBST, 0h 10m 0s (wash 1/3).

9.2.

Wash sections in TBST, 0h 10m 0s (wash 2/3).

9.3.

Wash sections in TBST, 0h 10m 0s (wash 3/3).

10.

Incubate sections in ABC, 0h 30m 0s at 4Room temperature.

11.

Prepare DAB solution (Sigma FAST, DAB Peroxidase Substrate Tablet Set), 1 DAB and 1 Urea Hydogen Peroxide tablet in 15mL distilled water.

12.

Wash sections in TBST, 10 min × 3.

12.1.

Wash sections in TBST, 0h 10m 0s (wash 1/3).

12.2.

Wash sections in TBST, 0h 10m 0s (wash 2/3).

12.3.

Wash sections in TBST, 0h 10m 0s (wash 3/3).

13.

Incubate sections in 1mL DAB, 0h 1m 0s-0h 2m 0s.

14.

Wash sections in TBST, 5 min × 3.

14.1.

Wash sections in TBST, 0h 5m 0s (wash 1/3).

14.2.

Wash sections in TBST, 0h 5m 0s (wash 2/3).

14.3.

Wash sections in TBST, 0h 5m 0s (wash 3/3).

15.

Mount the sections on positive charged plus slides.

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TH DAB staining

Abstract

Attachments

Steps

TH DAB staining

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