TITLE: 3,3-Diaminobenzidine Tetrahydrochloride (DAB) Immunohistochemistry on Mouse Brain Tissue Sections
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
This protocol details the staining for free floating mouse brain sections.

[STEPS]
SECTION: Day 1
1. Staining protocol for 35μm free floating mouse brain sections.
SECTION: Day 1
2. Staining is performed in glass staining dishes (Pyrex 36754-60) using 8-section staining nets (Ted Pella 36154-64) .
SECTION: Day 1
3. Gently rock the plates during the washing and incubation steps.
SECTION: Day 1
4. Wash sections: 3 times (5 min per wash) in Tris Buffer Saline (TBS) at Room temperature.
SECTION: Day 1
4.1. Wash sections: Wash for 5 min in Tris Buffer Saline (TBS) at Room temperature(1/3).
SECTION: Day 1
4.2. Wash sections: Wash for 5 min in Tris Buffer Saline (TBS) at Room temperature(2/3).
SECTION: Day 1
4.3. Wash sections: Wash for 5 min in Tris Buffer Saline (TBS) at Room temperature(3/3).
SECTION: Day 1
5. Quenching: Incubate sections in Quenching Solution (0.3% H202 in Methanol) for 10 min at 4 °C .
SECTION: Day 1
6. Wash sections: 3 short rinses in TBS then 2 times 5min in TBS at Room temperature.
SECTION: Day 1
6.1. Then wash for 5 min in TBS at Room temperature(1/2).
SECTION: Day 1
6.2. Then wash for 5 min in TBS at Room temperature(2/2).
SECTION: Day 1
7. Block sections in blocking solution (10% Normal Goat Serum (NGS) + 0.1% Triton-X in TBS) for 60 min at Room temperature.

SECTION: Day 1
8. Primary Antibodies: Dilute antibodies in 5% NGS + 0.1% Triton-X in TBS.
SECTION: Day 4
9. Wash 3 times (10 min each wash) in TBST (0.1% Tween 20 in TBS) at Room temperature.
SECTION: Day 4
9.1. Wash for 10 minin TBST (0.1%Tween 20 in TBS) at Room temperature(1/3).
SECTION: Day 4
9.2. Wash for 10 minin TBST (0.1%Tween 20 in TBS) at Room temperature(2/3).
SECTION: Day 4
9.3. Wash for 10 minin TBST (0.1%Tween 20 in TBS) at Room temperature(3/3).
SECTION: Day 4
10. Secondary Antibodies: 
SECTION: Day 4
11. Wash sections 3 times (10min each wash) in TBST at Room temperature.
SECTION: Day 4
11.1. Wash for 10 minin TBST at Room temperature(1/3).
SECTION: Day 4
11.2. Wash for 10 minin TBST at Room temperature(2/3).
SECTION: Day 4
11.3. Wash for 10 minin TBST at Room temperature(3/3).
SECTION: Day 4
12. Prepare ABC solution using VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) (PK-6100).
SECTION: Day 4
13. Incubate sections in ABC solution for 60 min at Room temperature.

SECTION: Day 4
14. Wash sections 3 times (10min each wash) in TBST at Room temperature.
SECTION: Day 4
14.1. Wash for 10 minin TBST at Room temperature(1/3).
SECTION: Day 4
14.2. Wash for 10 minin TBST at Room temperature(2/3).
SECTION: Day 4
14.3. Wash for 10 minin TBST at Room temperature(3/3).
SECTION: Day 4
15. Wash sections 2 times (5 min each wash) in TBS at Room temperature.
SECTION: Day 4
15.1. Wash sections for 5 min in TBS at Room temperature(1/2).
SECTION: Day 4
15.2. Wash sections for 5 min in TBS at Room temperature(2/2).
SECTION: Day 4
16. Prepare DAB solution using DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3'-diaminobenzidine) (SK-4100).

5 mL TBS + 2 drops of Reagent 1 (Buffer pH=7.5) + 4 drops Reagent 2 (DAB) + 2 drops Reagent 3 (H2O2).
Vortex between reagents (after reagent 1, after reagent 2 and after reagent 3).
Make 30 mL DAB solution per dish.
SECTION: Day 4
17. Revelation with DAB chromogen (until cells are visible):

 
All dishes must be developed for the same amount of time – do them together, ask a friend for help if you have more than 2 dishes of tissue.
SECTION: Day 4
18. Perform 3 short rinses in TBS at Room temperature.
SECTION: Day 4
18.1. Perform rinse in TBS at Room temperature(1/3).
SECTION: Day 4
18.2. Perform rinse in TBS at Room temperature(2/3).
SECTION: Day 4
18.3. Perform rinse in TBS at Room temperature(3/3).
SECTION: Day 4
19. Wash sections 5 time (5 min each wash) in TBS at Room temperature.
SECTION: Day 4
19.1. Wash sections for 5 min in TBS at Room temperature(1/5).
SECTION: Day 4
19.2. Wash sections for 5 min in TBS at Room temperature(2/5).
SECTION: Day 4
19.3. Wash sections for 5 min in TBS at Room temperature(3/5).
SECTION: Day 4
19.4. Wash sections for 5 min in TBS at Room temperature(4/5).
SECTION: Day 4
19.5. Wash sections for 5 min in TBS at Room temperature(5/5).
SECTION: Day 4
20. Mount sections:
SECTION: Day 5 or 6
21. Rehydrate slides in H2O for 2 min-3 min at Room temperature.

SECTION: Day 5 or 6
22. Dehydrate slides in sequential EtOH washes (70%, 95%, 100% EtOH) for 5 min each wash at Room temperature.
SECTION: Day 5 or 6
23. Clear tissue for 5 min in fresh Xylene I then incubate in fresh Xylene for ≥ 5 min.

a.	Keep slides in Xylene until coversliping.
b.	Perform Xylene steps and coversliping in a chemical fume hood.
SECTION: Day 5 or 6
24. Use Entellan mounting medium (Electron Microscopy Sciences 14802) to coverslip slides.
SECTION: Optional Cresyl Violet Protocol (NISSL stain)
25. After rehydration and before ethanol steps (dehydration).
SECTION: Optional Cresyl Violet Protocol (NISSL stain)
26. Incubate slides in fresh cresyl violet for 10 min at Room temperature.

SECTION: Optional Cresyl Violet Protocol (NISSL stain)
27. Proceed with dehydration steps normally.
SECTION: Optional Cresyl Violet Protocol (NISSL stain)
28. Must stain with cresyl violet for TH stereology in substantia nigra.
SECTION: Secondary Antibodies
29.  
 
 
SECTION: Day 1
2.1. The sections can be transferred between wells using a paint brush.
SECTION: Day 1
2.2. Volume of solution:

20 mL-30 mL for antibodies or ABC mix .
50 mL for washing.
SECTION: Day 1
8.1. Incubate in primary antibody solution for 2880 min at 4 °C.
SECTION: Day 1
8.2. Cover dishes with parafilm for this step.
SECTION: Day 4
10.1. Dilute secondary antibodies in TBST.
SECTION: Day 4
10.2. Biotinylated goat anti-rabbit IgG (1:500 dilution) or Biotinylated goat anti-mouse IgG (1:500 dilution).
SECTION: Day 4
10.3. Incubate for 120 min atRoom temperature or 1440 min at 4 °C.

SECTION: Day 4
12.1. ABC mix: 5 mL TBS + 2 drops of reagent A (vortex) + 2 drops reagent B (vortex) let stand at Room temperature for 30 min before using.
SECTION: Day 4
12.2. Vortex between reagents (after adding reagent A and after adding reagent B).
SECTION: Day 4
12.3. Make 30 mL ABC solution per dish.
SECTION: Day 4
20.1. Use a petri dish filled with PBS to mount the sections on superfrost plus slides (Fisher Scientific 12-550-15).

This is easiest to do using a medium sized paint brush.
SECTION: Day 4
20.2. After mounting let slides dry at Room temperature (2 min-10 min) then dip in miliQ H2O.

SECTION: Day 4
20.3. Dry slides 5 min at Room temperature before moving to the next step.
SECTION: Day 5 or 6
24.1. Apply one line of Entellan across the middle of the slide.
SECTION: Day 5 or 6
24.2. Cover with glass coverslip and gently press down with forceps.
SECTION: Day 5 or 6
24.3. Let slides dry in the fume hood for 60 min – then dry on the bench 5 min at Room temperature.
SECTION: Day 5 or 6
24.4. Gently remove excess Entellen with a razor blade.