DAB Immunohistochemistry (IHC) Staining for Stereological Analysis

Basic protocol for a peroxidase reaction using free floating sections.

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (use cell strainers for all washing steps).

Wash in 0.01Molarity (M) PBS containing 0.9% H202 ₂0₂ for 0h 10m 0s (90µL in 10mL PBS 0.01M) (Blocking of endogenous peroxidase).

Wash in 0.01Molarity (M)PBS (3x 10 min).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (1/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (2/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (3/3).

Incubate in primary antibodies for @ under stiring 48h 0m 0s @ 4°C under stiring.

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

• TH Rabbit – 1:1000

• GFP Rabbit 1:5000

• 5-HT Rabbit 1:1000

Wash in 0.01Molarity (M)PBS (3x 10 min).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (1/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (2/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (3/3).

Incubate in biotinylated secondary antibodies for at under stiring 12h 0m 0s 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).

Wash in 0.01Molarity (M)PBS (3x 10 min).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (1/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (2/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (3/3).

Incubate in streptavidin horseradish peroxidase for @ under stiring. 3h 0m 0s @ Room temperature under stiring.

• Diluted to 1:200 in 0.01Molarity (M) PBS containing 0.3% Triton X-100

Wash in 0.01Molarity (M)PBS (3x 10 min).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (1/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (2/3).

Wash in 0.01Molarity (M) PBS for 0h 10m 0s (3/3).

Prepare a reaction mixture which contains:

A	B
0.2M acetate buffer	5 mL
Nickel ammonium sulfate	250 mg
β-D-Glucose	20 mg
Ammonium chloride	4 mg

For 0.2 M Acetate Buffer pH 6.0

• Prepare 1L of 0.2Molarity (M) sodium acetate (27.216g for 1L)
• Prepare 1L of 0.2 acetic acid (11.5mL glacial acetic acid and top up to 1L with H₂Odd)
• Mix 900mL of 0.2Molarity (M) sodium acetate and 51.7mL of 0.2 acetic acid
• Top up to 1L with H₂O dd.
• Check the pH (Should be 6).

Prepare DAB solution (10mg/ml); 1 pill (10 mg) of DAB (-20°C) in 1 ml aliquot of ddH₂O. Vortex for 0h 1m 0s until the pill is dissolved.

Prepare Glucose oxidase (1mg/mL)

Glucose oxidase stock k:

• 2mg of glucose oxidase
• 2mL of acetate buffer 50millimolar (mM)
• Make aliquots of 80µL in advance .
• Keep the powder and aliquots at -20°C.

Add 250µL of DAB (10mg/1mL); for 10 mL reaction mixture 1mL for 10 mL reaction mixture) and 20µL of glucose oxidase for every 2.5 mL of mixture (1mg/mL); for 10 mL reaction mixture 80µL for 10 mL reaction mixture) to 10mL of reaction mixture.

Transfer the DAB reaction mixture in 10 ml syringe with 0.2 µm filter and place 1mL of DAB reaction mixture per well ( Use a 12-wells plate ).

Rinse sections in acetate buffer 0.1Molarity (M) acetate buffer for 0h 1m 0s.

For 0.1 M Acetate Buffer pH 6.0

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

Transfer sections to multi-wells containing DAB reaction mixture.

Wait 0h 0m 30s-0h 10m 0s for sections to develop a dark blue/purple nuclear stain under stirring (for TH, 45s is sufficient)

Remove immediately the sections from DAB reaction mixture.

Wash in 0.1Molarity (M) acetate buffer for 0h 10m 0s (it will stop reaction).

Mount sections on charged slides in 0.1M acetate buffer.

Allow slides to dry 48h 0m 0s-96h 0m 0s .

Defat slides in a series of ethanol baths 50-100% and then 0h 2m 0s in Xylene.

• 0h 2m 0s H₂O

•0h 2m 0s Cresyl Violet 0h 30m 0s at37°C before staining)

0h 1m 0s ₂H2O 0h 1m 0s• ₂ H2O0h 1m 0s • EtOH 500h 1m 0s

• EtOH 70h 1m 0s%

• EtOH 90%

• 10 dips EtOH0h 1m 0s90%

• EtOH0h 1m 0s100%

• Isopr0h 2m 0spanol

• 0h 2m 0sXylene

• Xylene

Cover with Permount under chemical hood.

Let dry 48h 0m 0s-72h 0m 0s under hood.