Monday, May 03, 2004

Description
Protocol for Metal Enhanced DAB Staining using HRP 2 Antibodies

Procedure
1. Carry out the fixation, permeabilization, and primary antibody incubation steps exactly as described above for the FITC detection method. You should carry out a separate dilution series of the 1 antibody to optimize its concentration for DAB detection: I ended up using 4-fold less 1 antibody for DAB than for FITC detection.

2. For the remainder of the procedure, solutions will be in PBST lacking sodium azide and lacking EDTA. (Azide inhibits HRP, and I left the EDTA so as not to chelate the metal ions in the developing solution).

3. After the 1 antibody incubation, wash 4X25 min. in PBST-B (-azide-EDTA) at room temp on a rotator.

4. Incubate 2 hours at room temperature in 20 l 2 antibody diluted in PBST-A(-azide-EDTA), agitating occasionally. I've been using a 1:60 dilution of HRP conjugated goat anti-rabbit IgG purchased from Biorad.

5. Wash the worms 4 times for 25 minutes each on a rotator at room temperature in

PBST-B (-azide-EDTA).

6. Development. Wear gloves and be careful here; DAB is a suspected carcinogen. I've been using a commercial DAB preparation from Pierce to develop the stain ("ImmunoPure Metal Enhanced DAB substrate Kit", catalog #34065). Store the 10X DAB solution at -20, and the 1X peroxide solution at 4. Just before use, mix the DAB solution to resuspend the metals, and combine 1 part DAB solution with 9 parts peroxide solution. Spin the woms down and remove as much supernatant as possible. Add 400 l of DAB/peroxide developing solution, and incubate on a rotator at room temp. 1-20 minutes. Can remove an aliquot of the developing worms to a glass depression slide and watch them develop under a dissecting scope to decide when to stop the reaction. Most of the staining occurs very quickly (1-2 minutes), and very little occurs after that. I've routinely been developing for 8 minutes.

7. To stop the reaction: spin the worms down, remove the supernatant. Wash 1 min. on a rotator in PBST-B(-azide-EDTA), followed by three more 5 minute washes.

8. Mount 3 l stained worms, with 3 l 80% glycerol, under a 18 mm square coverslip sealed with nail polish. The stained worms are stable in the fridge for at least a week.

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