TITLE: DAB Staining for GFP on Free-floating Fixed NHP Brain Tissue  
AUTHORS: Aydin Alikaya, William Stauffer

[DESCRIPTION]
This protocol details the procedure for immunohistochemical 3,3’-Diaminobenzidine (DAB) staining of free-floating fixed brain tissue sections using the avidin/biotin ABC complex to detect Green Fluorescence Protein (GFP).  

This protocol has been tested with free-floating non-human primate (NHP) brain tissue that has been fixed with 4% paraformaldehyde, cryoprotected with sucrose gradients, and cryo-sectioned  . 


[GUIDELINES]
When using 6 well tissue culture plates [Falcon, 353046] to react individual sections, you will need 2+ mL solutions for each well plate. 

When using circular staining nets [e.g., Brain Research Laboratories #4115] to react multiple series of sections, you will need 50 mL solutions for each staining net. 

[STEPS]
SECTION: Part I (Day 1)
1. Bring tissue to Room temperature in Phosphate Buffered Saline (PBS, pH 7.2-7.4) on an orbital shaker for 30 minutes. 30 min

SECTION: Part I (Day 1)
2. Prepare Peroxide Solution (0.3 % H2O2) in dH2O.
For 10 mL 0.3% H2O2 use:
100 µL 30% H2O2 
9900 µL dH2O


SECTION: Part I (Day 1)
3. Prepare Normal Goat Serum Blocking Solution in PBS.
To 10 mL PBS add: 
150 µL Normal Goat Serum (= 3 drops of serum from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001)

SECTION: Part I (Day 1)
4. Prepare Primary Antibody Solution (rabbit anti-GFP) at 1:10000 dilution in PBS: 
1 µL rabbit anti-GFP (Thermo Fisher, Molecular Probes Cat# A11122, RRID:AB_221569)
9999 µL PBS. 
SECTION: Part I (Day 1)
5. Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 5 min

SECTION: Part I (Day 1)
6. Quench endogenous peroxide in Peroxide Solution (0.3 H2O2) on a shaker at Room temperature: 30 minutes. 30 min

SECTION: Part I (Day 1)
7. Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 5 min

SECTION: Part I (Day 1)
8. Incubate in  Normal Goat Serum Blocking 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 5 min.

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

SECTION: Part II (Day 2)
11. Prepare ABC Solution in PBS (at least 30 minutes before use). 30 min 
To 10 mL PBS add:
2 drops A from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001.
2 drops B from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001.
SECTION: Part II (Day 2)
12. Prepare Secondary Antibody Solution (1:200) in PBS.
To 10 mL PBS add:
150 µL (= 3 drops from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001) of Normal Goat Serum. 
50 µL (= 1 drop from Vectastain ABC-HRP Kit, Peroxidase Rabbit IgG PK-4001) biotinylated goat anti-rabbit IgG secondary antibody.


SECTION: Part II (Day 2)
13. Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 5 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 on a shaker at Room temperature: 3 x 5 minutes. 5 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 on a shaker at Room temperature: 3 x 5 minutes. 5 min
SECTION: Part II (Day 2)
18. Prepare DAB 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 DAB Peroxide Substrate Solution on a shaker at Room temperature:
3 min -6 min. 

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

SECTION: Part II (Day 2)
20. Rinse in PBS on a shaker at Room temperature: 3 x 5 minutes. 5 min

SECTION: Part II (Day 2)
21. Mount tissue on charged glass slides 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).