Aug 27, 2024
DAB Staining for Tyrosine Hydroxylase (TH) on Free-floating Fixed NHP Brain Tissue
- 1Department of Neurobiology, University of Pittsburgh
Protocol Citation: Andreea Bostan 2024. DAB Staining for Tyrosine Hydroxylase (TH) on Free-floating Fixed NHP Brain Tissue . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2q1dql1y/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: August 27, 2024
Last Modified: August 27, 2024
Protocol Integer ID: 106519
Keywords: ASAPCRN, Immunostaining, DAB, NHP Brain Tissue, TH
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-020519
Abstract
This protocol details the procedure for immunohistochemical 3,3’-Diaminobenzidine (DAB) staining for Tyrosine Hydroxilase (TH) of free-floating fixed brain tissue sections using the avidin/biotin ABC complex and the Rabbit Polyclonal Anti-TH NB300-109 (Novus).
This protocol has been tested with free-floating non-human primate (NHP) brain tissue that has been fixed (10% formalin or 4% paraformaldehyde), cryoprotected (sucrose or glycerol gradients), and cryo-sectioned 50 µm .
Guidelines
This protocol has been optimized for using 6 well tissue culture plates [Falcon, 353046] to react individual sections. You will need 2+ mL solutions for each well plate.
It is recommended to use new culture plates for antibody incubation and discard plates used for DAB.
Plates used for other steps can be washed and re-used.
Materials
Tissue:
Brain tissue sections (20 - 50 μm).
Materials/Equipment:
- Tissue culture plates or circular staining nets
- Orbital shaker
- Fume hood
- Nitrile Gloves
- Glass slides (charged or subbed)
Reagents:
- Phosphate-buffered saline (PBS)
- Phosphate-buffered saline (PBS) with 0.2% Triton-X
- Akoya Blocking Reagent Powder (SKU FP1012) to make TNB Blocking Buffer/
- Hydrogen Peroxide: H2O2 (3% or 30%)
- Distilled water: dH2O
- Primary Antibody: Novus Biologicals Tyrosine Hydroxylase (TH) Polyclonal Rabbit.
- Secondary Antibody (to match the host of the primary antibody): biotinylated Goat-Anit Rabbit.
- Normal Serum Blocking Solution (e.g., Normal Goat Serum, S-1000-20)
- Vectastain Elite ABC Peroxidase Kit (Standard) (PK-6100) (Vector Laboratories)
- Vectastain ABC-HRP Kit, Peroxidase (Rabbit IgG) (PK-4001, Vector Laboratories)
- DAB Substrate Kit
Examples:
Peroxidase (HRP) with Nickel (3,3'-diaminobenzidine) (SK-4100) (Vector Laboratories)
ImmPACT DAB (SK-4105)
Safety warnings
Use appropriate care when using hydrogen peroxide (reactive, can cause skin/eye damage) and DAB (suspected carcinogen). Collect DAB solution for chemical waste disposal.
Part I (Day 1)
Part I (Day 1)
3h
3h
Bring tissue to Room temperature in phosphate-buffered saline (PBS) on an orbital shaker for 30 minutes.
00:30:00 .
30m
Prepare Peroxide Solution (3 % H2O2) in dH2O.
E.g., for 10 mL 3% H2O2 use:
- 1000 µL 30% H2O2
- 9000 µL dH2O
5m
Prepare Blocking Serum Solution: Normal Goat Serum (NGS) in TNB Blocking Buffer.
E.g., in 10 mL buffer (TNB Blocking Buffer) add:
- 150 µL NGS (or 3 drops of normal serum if using an ABC kit, e.g. Vectastain ABC-HRP Kit Rabbit IgG PK-4001)
5m
Prepare Primary Antibody Solution: anti-TH NB300-109 at 1:1000 TNB Blocking Buffer.
E.g., for 10 mL Primary Antibody Solution use:
- 9990 µL TNB Blocking Buffer
- 10 µL anti-TH NB300-109
5m
Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker at Room temperature :
3 x 3 minutes. 00:09:00
9m
Quench endogenous peroxide in Peroxide Solution (3 % H2O2) on a shaker atRoom temperature :
2 x 10 - 15 minutes. 00:20:00 -01:00:00
1h 20m
Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker at Room temperature :
3 x 3 minutes. 00:09:00
9m
Incubate in Blocking Serum Solution on a shaker at RT: 1 hour.
01:00:00 .
DO NOT RINSE after blocking serum.
1h
Incubate in Primary Antibody Solution on a shaker at 4 °C : overnight x 3 (60 - 72 hours).
72:00:00 .
3d
Part II (Day 2)
Part II (Day 2)
4h
4h
Bring tissue (in the Primary Antibody Solution) to Room temperature on a shaker (45 minutes). 00:30:00 -01:00:00
30m
Prepare ABC Solution in PBS with 0.2% Triton X (PBS-Tx) (at least 30 minutes before use). 00:30:00 .
5m
Prepare Secondary Antibody Solution (1:200) in TNB Blocking Buffer.
In 10 mL TNB Blocking Buffer add:
- 150 µL Normal Goat Serum (NGS) (= 3 drops of normal serum if using an ABC kit, e.g. Vectastain ABC-HRP Kit Rabbit IgG PK-4001).
- 50 µL Secondary biotinylated goat-anti rabbit (= 1 drop of normal goat serum if using an ABC kit, e.g. Vectastain ABC-HRP Kit Rabbit IgG PK-4001).
5m
Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker atRoom temperature :
3 x 3 minutes. 00:09:00 .
9m
Incubate in Secondary Antibody Solution on a shaker at Room temperature :
30 minutes. 00:30:00 .
30m
Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker atRoom temperature :
3 x 3 minutes. 00:09:00 .
9m
Incubate in ABC Solution on a shaker at Room temperature :
60 minutes. 01:00:00 .
1h
Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker atRoom temperature :
3 x 3 minutes. 00:09:00 .
9m
Prepare Peroxide Substrate Solution in dH2O.
To use the Vector Labs DAB Peroxidase Substrate Kit (SK-4100):
In 5 mL dH2O:
- 2 drops Reagent 1
- 4 drops Reagent 2
- 2 drops Reagent 3
- [optional] 2 drops of Reagent 4 (Nickel) if a black reaction product is desired
Note: Mix well before use. Use immediately.
5m
Incubate in Peroxide Substrate Solution on a shaker at Room temperature :
3 - 10 minutes. 00:03:00 -00:06:00 .
Note: Watch the tissue closely to avoid high background staining.
6m
Rinse in buffer (e.g. PBS) on a shaker at Room temperature :
3 x 3 minutes. 00:09:00 .
9m
Mount tissue on glass slides (subbed or charged) in 1:8 PBS in dH2O and let air dry.
Rinse slides with dH2O and let air dry (preferably in a hood).
Coverslip clean and dry slides with Cytoseal 60 (Thermo Fisher #830-16).