For 40-50µm vibratome sections.

**DAB can be combined with Cresyl Violet or Heamotoxylin as counterstains.

Method:

Tissue Preparation

1. 

   Fix mouse embryos with 4%PFA or 2.5% Acrolein/4% PFA in PBS either by immersion fixation (<E14) or transcardial perfusion (>E15)

2. Dissect fixed mouse brains and embed in 3% Noble Agar
3. Cut floating sections on vibratome at 45-50µm thickness in PBS
4. Store sections short term in PBS or long term in 0.02% Sodium Azide in PBS at 4ºC

Blocking and Primary Antibody (DAY 1)

**All steps are at room temperature on a lab rotator**

1. Put desired sections into net wells – If brains were fixed in acrolein start at step 2, for PFA fixed brains start at step 3.
2. Wash acrolein-fixed brains in 1% sodium borohydride in PBS for 20 mins. Stay as close to the time as possible (too much will ruin your sections, too little will effect the level of background)
3. Wash in PBS for 10 mins (x3)
4. Black sections with 0.2% Triton-X 100/2% normal serum in PBS for 2 hrs (optional 2% BSA). **Serum should be from species of the secondary antibody (e.g. Goat anti-rabbit 1º antibody should be blocked in normal goat serum). Make enough blocking solution for 2 runs (it is used to block and the for the 1ºantibody step)
5. Incubate with 1º antibody diluted to the desired concentration in blocking solution overnight.

Secondary Antibody, Amplification and Color Reaction (DAY 2)

1. Wash in PBS for 20 mins (x3)
2. Incubate in 2º antibody diluted to the desired concentration in 0.2% Triton-X 100 in PBS for 1 hour.
3. Wash in PBS for 20 mins (x3). (Make up AB complex during first wash and keep on ice/in fridge until used).
4. Incubate for 1 hour in AB complex made up 1 hour before use as: Solution A 1:500/Solution B (1:500)/0.2% Triton-x 100 in PBS
5. Wash in PBS for 10 mins (x3)
6. Dissolve 1.25g of nickel sulphate in 50mL of 0.175M sodium acetate, i.e. 5mL of 1.75M sodium acetate in 45 milliQ H2O
7. Add 1xDAB tablet (10mg) per 50mL nickel sulphate solution. Parafilm and sonicate until dissolved. It is critical that every step involving DAB is done in the fume hood as DAB is carcinogenic and teratogenic
8. Filter through filter paper in aluminium foil tunnel into a new 50mL tube
9. Add 5µL of 30% hydrogen peroxide to the DAB-Nickel sulphate solution to start the reaction. **Horseradish peroxidase of biotinylated avidin reacts + H202 = oxidizes DAB on that sites of the ligand to form a brown insoluble precipitate.
10. Transfer DAB solution to new plates by plastic transfer pipette
11. Drain net wells of PBS throughly before placing in DAB solution
12. Cover plate and slowly swirl plate by hand, to allow maximum exposure to sections
13. Start timer. Reaction shouldn’t take longer than 20 minutes
14. Drain thoroughly of DAB before replacing in PBS to stop reaction
15. Wash in PBS for 5 minutes (x3)
16. To clean up DAB reaction, immerse plates in bleach then soak plates in detergent overnight. Wash throughly and rinse the water the following day
17. Mount sections onto slides by floating in PBS. Allow at least 1 hour to dry.
18.  Haematoxylin Counterstain if necessary here.
19. Dehydrate in the fume hood using the following sequence:
1. 1x2min washes of the following:dH20,75% EtOH; 95% EtOH.
2. Followed by 2x2min washes in 100% EtOH,
3. Then 3x2min washes in 100% Histolene (Xylene may be substituted here)
20. Use DPX (xylene-based mounting media) to coverslip, leave in fume hood to dry overnight.
21. Store slides in slide boxes at room temperature

DAB stained neurons with
Haemotoxylin counterstain