TITLE: DAB Immunohistochemistry (IHC) Staining for Stereological Analysis
AUTHORS: Nicolas Giguère, louis-eric.trudeau Trudeau

[DESCRIPTION]
DAB (3,3'-diaminobenzidine) is oxidized in the presence of peroxidase and hydrogen peroxide resulting in the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity.DAB (3, 3'-diaminobenzidine) produces a dark brown reaction product and can be used for immunohistochemical and applications. This protocol details the DAB immunohistochemistry staining for stereological analysis of 40 µM slices cut with cryostat and stored in antifreeze.

[GUIDELINES]
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee

[STEPS]
SECTION: Procedures
1. Basic protocol for a peroxidase reaction using free floating sections. 

 

Wash in 0.01 Molarity (M) PBS for 10 min (use cell strainers for all washing steps).
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2. Wash in 0.01 Molarity (M) PBS containing 0.9% H202 for 10 min (90 µL in 10 mL PBS 0.01M) (Blocking of endogenous peroxidase).
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3. Wash in 0.01 Molarity (M)PBS (3x 10 min).
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3.1. Wash in 0.01 Molarity (M) PBS for 10 min (1/3).
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3.2. Wash in 0.01 Molarity (M) PBS for 10 min (2/3).
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3.3. Wash in 0.01 Molarity (M) PBS for 10 min (3/3).
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4. Incubate in primary antibodies for 2880 min @ 4 °C under stiring.

Diluted to its optimal titer (1:1000-1:10,000) in 0.01 Molarity (M) containing 0.3% Triton X-100

 

• TH Rabbit – 1:1000
• GFP Rabbit 1:5000
• 5-HT Rabbit 1:1000
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6. Incubate in biotinylated secondary antibodies for 720 min at 4 °C under stiring.

• Diluted to 1:200 in 0.01 M PBS containing 0.3% Trition X-100 (this antibody is stored in 1:2 glycerol, consequently the concentration will be 1:100).
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7. Wash in 0.01 Molarity (M)PBS (3x 10 min).
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7.1. Wash in 0.01 Molarity (M) PBS for 10 min (1/3).
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7.2. Wash in 0.01 Molarity (M) PBS for 10 min (2/3).
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7.3. Wash in 0.01 Molarity (M) PBS for 10 min (3/3).
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8. Incubate in streptavidin horseradish peroxidase for 180 min @ Room temperature under stiring.

• Diluted to 1:200 in 0.01 Molarity (M) PBS containing 0.3% Triton X-100
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5. Wash in 0.01 Molarity (M)PBS (3x 10 min).
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5.1. Wash in 0.01 Molarity (M) PBS for 10 min (1/3).
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5.2. Wash in 0.01 Molarity (M) PBS for 10 min (2/3).
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5.3. Wash in 0.01 Molarity (M) PBS for 10 min (3/3).
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10. Prepare a reaction mixture which contains:
 
 
 
 


For 0.2 M Acetate Buffer pH 6.0
 
Prepare 1 L of 0.2 Molarity (M) sodium acetate (27.216 g for 1L)
Prepare 1 L of  0.2 3261  acetic acid (11.5 mL glacial acetic acid and top up to 1L with H2Odd)
Mix 900 mL of 0.2 Molarity (M) sodium acetate and 51.7 mL of 0.2 3261 acetic acid 
Top up to 1 L with H2O dd.
Check the pH (Should be pH 6).
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11. Prepare DAB solution (10mg/ml); 1 pill (10 mg) of DAB (-20 °C) in 1 ml aliquot of ddH2O. Vortex for 1 min until the pill is dissolved.

 

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12. Prepare Glucose oxidase (1 mg/mL)

Glucose oxidase stock:

2 mg of glucose oxidase
2 mL of acetate buffer 50 millimolar (mM)
Make aliquots of 80 µL in advance .
Keep the powder and aliquots at -20 °C.
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13. Add 250 µL of DAB (10mg/1mL); 1 mL for 10 mL reaction mixture) and 20 µL of glucose oxidase for every 2.5 mL of mixture (1mg/mL); 80 µL for 10 mL reaction mixture) to 10 mL of reaction mixture.
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9. Wash in 0.01 Molarity (M)PBS (3x 10 min).
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9.1. Wash in 0.01 Molarity (M) PBS for 10 min (1/3).
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9.2. Wash in 0.01 Molarity (M) PBS for 10 min (2/3).
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9.3. Wash in 0.01 Molarity (M) PBS for 10 min (3/3).
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14. Transfer the DAB reaction mixture in 10 ml syringe with 0.2 µm filter and place 1 mL of DAB reaction mixture per well (Use a 12-wells plate).
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15. Rinse sections in 0.1 Molarity (M) acetate buffer for 1 min.

For 0.1 M Acetate Buffer pH 6.0 

Mix 500 mL of 0.2 Molarity (M) Acetate Buffer and 500 mLof H2Odd.

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16. Transfer sections to multi-wells containing DAB reaction mixture.
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17. Wait 30 s-10 min for sections to develop a dark blue/purple nuclear stain under stirring (for TH, 45s is sufficient)

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18. Remove immediately the sections from DAB reaction mixture.
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19. Wash in 0.1 Molarity (M) acetate buffer for 10 min (it will stop reaction).

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20. Mount sections on charged slides in 0.1M acetate buffer.
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21. Allow slides to dry 2880 min-5760 min .

 


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22. Defat slides in a series of ethanol baths 50-100% and then 2 min in Xylene. 

 

• 2 min H2O
• 2 min Cresyl Violet (30 min at 37 °C before staining)
• 1 min H2O
• 1 min H2O
• 1 min EtOH 50%
• 1 min EtOH 70%
• 1 minEtOH 90%
• 10 dips EtOH 90%
• 1 min EtOH 100%
• 1 min Isopropanol
• 2 min Xylene
• 2 min Xylene

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23. Cover with Permount under chemical hood.
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24. Let dry 2880 min-4320 min under hood.