TITLE: Standard DAB Staining for Free-floating Fixed NHP Brain Tissue  
AUTHORS: Andreea C. Bostan

[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. 

This protocol has been tested with free-floating non-human primate (NHP) and rodent (mouse, rat) brain tissue that has been fixed (10% formalin or 4% paraformaldehyde), cryoprotected (sucrose or glycerol 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. 

[STEPS]
SECTION: Part I (Day 1)
1. Bring tissue to Room temperature in buffer (e g., Phosphate buffered saline, PBS) on an orbital shaker for 30 minutes. 30 min. 

SECTION: Part I (Day 1)
2. Prepare Peroxide Solution (0.3 - 3 % H2O2) in dH2O.
E.g., for 10 mL 0.3% H2O2 use:
100 µL 30% H2O2 
9900 µL dH2O


SECTION: Part I (Day 1)
3. Prepare Blocking Serum Solution (e.g. Normal Horse, Normal Goat Serum) using a serum that matches the host of the secondary antibody (e.g. Normal Horse Serum for a Horse anti-Mouse secondary, Normal Goat Serum for a Goat anti-Rabbit secondary). 
E.g., in 10 mL buffer (PBS) add: 
150 µL normal serum (or 3 drops of normal serum if using an ABC kit, e.g. Vectastain ABC-HRP Kit, Peroxidase Mouse IgG PK-4002, Rabbit IgG PK-4001)

SECTION: Part I (Day 1)
4. Prepare Primary Antibody Solution at the appropriate dilution in buffer (e.g., 1:1000 in PBS).
SECTION: Part I (Day 1)
5. Rinse in buffer (e.g. PBS) on a shaker at Room temperature: 3 x 3-5 minutes. 3 min -5 min

SECTION: Part I (Day 1)
6. Quench endogenous peroxide in Peroxide Solution (0.3 - 3 % H2O2) on a shaker atRoom temperature: 30 - 60 minutes. 30 min -60 min

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

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

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, or longer (20 - 72 hours depending on the antibody). 

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

SECTION: Part II (Day 2)
11. Prepare ABC Solution in buffer (e.g. PBS) (at least 30 minutes before use). 30 min . 

SECTION: Part II (Day 2)
12. Prepare Secondary Antibody Solution (1:200) in buffer (e.g. PBS). 
In 10 mL buffer add:
150 µL (= 3 drops of normal serum from a Vector Labs kit) of normal serum (matched to the host of your secondary antibody)
50 µL (= 1 drop secondary antibody from a Vector Labs kit) of biotinylated secondary antibody (matched to the host of your primary Antibody )


SECTION: Part II (Day 2)
13. Rinse in buffer (e.g. PBS) on a shaker atRoom temperature: 3 x 3-5 minutes. 3 min -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 buffer (e.g. PBS) on a shaker at Room temperature: 3 x 3-5 minutes. 3 min -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 buffer (e.g. PBS) on a shaker at Room temperature: 3 x 3-5 minutes. 3 min -5 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 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 3-5 minutes. 3 min -5 min.  

SECTION: Part II (Day 2)
21. Mount tissue on glass slides (subbed or charged) in 1:8 buffer 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).