Jul 15, 2022

Public workspaceTH DAB staining

DOI
dx.doi.org/10.17504/protocols.io.j8nlk4yw1g5r/v1
  • 1Department of Neurology Massachusetts General Hospital Harvard Medical School Charlestown MA 02129 USA;
  • 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network Chevy Chase MD 20815 USA;
  • 3Department of Neurology Longhua Hospital Shanghai University of Traditional Chinese Medicine Shanghai 200032 China
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DOI: dx.doi.org/10.17504/protocols.io.j8nlk4yw1g5r/v1
Protocol Citation: Pranay Srivastava, Waijiao Cai, Xiqun Chen 2022. TH DAB staining. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk4yw1g5r/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: June 16, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 50825
Keywords: TH DAB staining, TBST, ASAPCRN
Abstract
This protocol details the stating procedure for TH DAB staining.
Attachments
Icon for ds8ybjtc7.pdf
ds8ybjtc7.pdf
178KB
Materials
Materials:

ReagentTyrosine hydroxylase polyclonal antibodyEnzo Life SciencesCatalog #BML-SA497-0100
ReagentGoat Anti-Rabbit IgG Biotin ConjugateMillipore SigmaCatalog #OS03B
ReagentVECTASTAIN Elite ABC HRP Kit (Peroxidase, Standard)Vector LaboratoriesCatalog #PK-6100

TH DAB staining
TH DAB staining
6h 18m
6h 18m
Wash a full set of sections (6-8 sections) in a well in PBS, 5 min × 3.
Wash
Wash a full set of sections (6-8 sections) in a well in PBS, Duration00:05:00 (wash 1/3) (if stored in cryoprotective solution).

5m
Wash
Wash a full set of sections (6-8 sections) in a well in PBS, Duration00:05:00 (wash 2/3) (if stored in cryoprotective solution).
5m
Wash
Wash a full set of sections (6-8 sections) in a well in PBS, Duration00:05:00 (wash 3/3) (if stored in cryoprotective solution).
5m
Wash
Incubate sections in 1% H2O2 (Amount1 mL 30% H2O2 in Amount29 mL TBST), Duration00:30:00 at TemperatureRoom temperature .

30m
Incubation
Wash sections in TBST 5 min × 3.
Wash
Wash sections in TBST Duration00:05:00 (wash 1/3).
5m
Wash
Wash sections in TBST Duration00:05:00 (wash 2/3).
5m
Wash
Wash sections in TBST Duration00:05:00 (wash 3/3).
5m
Wash
Block sections in 5% NGS/TBST, Duration01:00:00 at TemperatureRoom temperature .

1h
Incubate sections in rabbit anti-TH (#SA-497), 1:3000 in TBST, Temperature4 °C DurationOvernight .

Incubation
Overnight
Wash sections in TBST, 10 min × 3.
Wash
Wash sections in TBST, Duration00:10:00 (wash 1/3).

10m
Wash
Wash sections in TBST, Duration00:10:00 (wash 2/3).

10m
Wash
Wash sections in TBST, Duration00:10:00 (wash 3/3).

10m
Wash
Incubate sections in anti-rabbit biotinylated (#OS03B), 1:3000, Duration01:00:00 at TemperatureRoom temperature .

1h
Incubation
Prepare ABC (Avidin-Biotin-Complex, Vectastain ABC Kit, # PK-6100): 1 drop A and 1 drop B in Amount2.5 mL TBST, at least 30 min before use.

Wash sections in TBST, 10 min × 3.
Wash
Wash sections in TBST, Duration00:10:00 (wash 1/3).

10m
Wash
Wash sections in TBST, Duration00:10:00 (wash 2/3).
10m
Wash
Wash sections in TBST, Duration00:10:00 (wash 3/3).
10m
Wash
Incubate sections in ABC, Duration00:30:00 at TemperatureRoom temperature .

30m
Incubation
Prepare DAB solution (Sigma FAST, DAB Peroxidase Substrate Tablet Set), 1 DAB and 1 Urea Hydogen Peroxide tablet in Amount15 mL distilled water.

Wash sections in TBST, 10 min × 3.
Wash
Wash sections in TBST, Duration00:10:00 (wash 1/3).

10m
Wash
Wash sections in TBST, Duration00:10:00 (wash 2/3).
10m
Wash
Wash sections in TBST, Duration00:10:00 (wash 3/3).
10m
Wash
Incubate sections in Amount1 mL DAB, Duration00:01:00 -Duration00:02:00 .

3m
Incubation
Wash sections in TBST, 5 min × 3.
Wash
Wash sections in TBST, Duration00:05:00 (wash 1/3).

5m
Wash
Wash sections in TBST, Duration00:05:00 (wash 2/3).
5m
Wash
Wash sections in TBST, Duration00:05:00 (wash 3/3).
5m
Wash
Mount the sections on positive charged plus slides.