TITLE: DAB Staining for Tyrosine Hydroxylase (TH) on Free-floating Fixed NHP Brain Tissue  
AUTHORS: Andreea Bostan

[DESCRIPTION]
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  . 


[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. 


[STEPS]
SECTION: Part I (Day 1)
1. Bring tissue to Room temperaturein phosphate-buffered saline (PBS) on an orbital shaker for 30 minutes. 
30 min. 

SECTION: Part I (Day 1)
2. Prepare Peroxide Solution (3 % H2O2) in dH2O.
E.g., for 10 mL 3% H2O2 use:
1000 µL30% H2O2
9000 µLdH2O
SECTION: Part I (Day 1)
3. 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)

SECTION: Part I (Day 1)
4. Prepare Primary Antibody Solution: anti-TH NB300-109 at 1:1000 TNB Blocking Buffer.
E.g.,  for 10 mL Primary Antibody Solution use: 
9990 µLTNB Blocking Buffer
10 µLanti-TH NB300-109
SECTION: Part I (Day 1)
5. Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker at Room temperature: 
3 x 3 minutes. 9 min
SECTION: Part I (Day 1)
6. Quench endogenous peroxide in Peroxide Solution (3 % H2O2) on a shaker atRoom temperature: 
2 x 10 - 15 minutes. 20 min -60 min

SECTION: Part I (Day 1)
7. Rinse in PBS with 0.2% Triton X (PBS-Tx) on a shaker at Room temperature: 
3 x 3 minutes. 9 min

SECTION: Part I (Day 1)
8. Incubate in Blocking Serum Solution on a shaker at RT: 1 hour. 
60 min . 

DO NOT RINSE after blocking serum. 
SECTION: Part I (Day 1)
9. Incubate in Primary Antibody Solution on a shaker at 4 °C: overnight x 3 (60 - 72 hours).
4320 min . 

SECTION: Part II (Day 2)
10. Bring tissue (in the Primary Antibody Solution) to Room temperature on a shaker (45 minutes). 30 min -60 min
 

SECTION: Part II (Day 2)
11. Prepare ABC Solution in PBS with 0.2% Triton X (PBS-Tx) (at least 30 minutes before use). 30 min . 

SECTION: Part II (Day 2)
12. Prepare Secondary Antibody Solution (1:200) in TNB Blocking Buffer.
In 10 mL TNB Blocking Buffer add:
150 µLNormal 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). 

SECTION: Part II (Day 2)
13. Rinse in  PBS with 0.2% Triton X (PBS-Tx) on a shaker atRoom temperature: 
3 x 3 minutes. 9 min.

SECTION: Part II (Day 2)
14. Incubate in Secondary Antibody Solution on a shaker at Room temperature: 
30 minutes. 30 min. 

SECTION: Part II (Day 2)
15. Rinse in  PBS with 0.2% Triton X (PBS-Tx) on a shaker atRoom temperature: 
3 x 3 minutes. 9 min.

SECTION: Part II (Day 2)
16. Incubate in ABC Solution on a shaker at Room temperature: 
60 minutes. 60 min. 

SECTION: Part II (Day 2)
17. Rinse in  PBS with 0.2% Triton X (PBS-Tx) on a shaker atRoom temperature: 
3 x 3 minutes. 9 min.

SECTION: Part II (Day 2)
18. 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. 

SECTION: Part II (Day 2)
19. Incubate in Peroxide Substrate Solution on a shaker at Room temperature: 
3 - 10 minutes. 3 min -6 min. 

Note: Watch the tissue closely to avoid high background staining. 

SECTION: Part II (Day 2)
20. Rinse in buffer (e.g. PBS) on a shaker at Room temperature: 
3 x 3 minutes. 9 min.

SECTION: Part II (Day 2)
21. Mount tissue on glass slides (subbed or charged) in 1:8 PBS in dH2O and let air dry. 
SECTION: Part II (Day 2)
22. Rinse slides with dH2O and let air dry (preferably in a hood).
SECTION: Part II (Day 2)
23. Coverslip clean and dry slides with Cytoseal 60 (Thermo Fisher #830-16).