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[old protocol needs revising]

For the 1° antibody, 2° antibody, and ABC solutions, mix 300 µl per well of 6 NTS sections, or 500 µl per well of 6 forebrain or PBN sections.

c-Fos Primary Antibody 1 part anti-c-Fos 1:1000 stock plus 2 parts PBS/BSA.

Incubate overnight (12 - 16 hrs) on the shaker table. Be sure to place wet papertowels in the bottom of the baking pan and seal the pan with Saran Wrap -- otherwise the buffer will evaporate and your tissue sections will be desicated...

anti-Sheep Secondary Antibody 5 µl anti-sheep biotinylated rabbit antibody in 1 ml PBS/BSA.

Incubate for 1 hr on the shaker table.

ABC Elite Solution 20 µl solution A and 20 µl solution B per 1 ml of PBS/BSA.

30 minutes before use (i.e. when the sections go into the first wash after the 1 hr secondary antibody incubation) mix 20 µl solution A and 20 µl solution B per 1 ml of PBS/BSA. Vortex, and allow to stand for 30 minutes, then pipette into the wells and transfer the tissue sections into the ABC. Incubate for 1 hr on the shaker table.

DAB Solution 12 mg DAB in 12 ml H20, add 12 ml 0.2 M PB, add 3 µl 30% H202.

On the analytical balance, measure out 12 mg DAB into a dark or foil-covered flask. Wear gloves, lab coat, and face mask, because DAB is a known carcinogen. Put spatula into a bleach, water, and alconox solution for decontamination (along with all other things that contact the DAB). Add 12 ml ddH20, cover with parafilm to prevent spillage, and dissolve by gently swirling the flask. The DAB should dissolve easily in water, and looking in the flask you should see no undissolved chunks but a light brown solution (it will turn browner with light exposure over the course of the experiment). Add 12 ml 0.2 M PB, to bring the concentration to 0.1 M PB (DAB will not dissolve in 0.1M PB, so it must be brought to this concentration after dissolving in water).

When ready to react the tissue, pipette 3 µl of 30% hydrogen peroxide into the DAB flask and swirl to mix. This will activate the peroxidase bound to the ABC complex (bound to the secondary antibody (bound to the primary antibody (bound to the c-Fos protein))).