Staining Sections With Antibodies Using PAP

(Source: R. BreMiller from Zebrafish Book 5th Edition)

1. To conserve the volume of solution needed for the staining reaction, circumscribe the sections with a ring of hydrophobic material. A PAP pen (Research Products International Corp) is very effective for this purpose

2. Vibratome sections may benefit from permeabilization by treating with 0.1% Triton in PBS/BSA/DMSO for 5 to 10 minutes.

3. Remove Triton solution if used and replace with 2% normal goat serum in PBS/BSA/DMSO for 20 minutes. A pipette tip connected by tubing to an aspirator facilitates removal of solutions from the sections

4. Replace blocking solution with primary antibody. Treat sections overnight at 4°C. To prevent sections from drying out, place slides in large plastic Petri dishes and cover the dishes with lids into which filter paper moistened with distilled water has been fitted.

5. Wash sections with PBS/BSA/DMSO 4 times for 5 minutes each.

6. Treat with secondary antibody diluted according to manufacturers recommendation with PBS/BSA/DMSO for 1 hour at room temperature.

7. Wash with PBS/BSA/DMSO as above.

8. Treat with peroxidase anti-peroxidase (PAP) diluted according to manufacturers recommendation with PBS/BSA/DMSO for 1 hour at room temperature.

9. Wash as above.

10. Repeat sequence with secondary antibody and PAP. For frozen sections, the secondary antibody and PAP can be used for 2 hours each, in which case the sequence is not repeated.

11. Wash with 0.1 M PO4 buffer, pH 7.3, several changes.

12. Stain with DAB pre-soak solution for 5 min. Plastic "mailers" make good disposable staining containers for five slides and hold 12-15 ml of solution.

13. Pour off the DAB solution. Add to it 3% H2O2 in an appropriate volume to give a final concentration of 0.004% and return the solution to the staining containers. Incubate, using a shaker if possible, until sections show color. In most cases, a staining time of 5 to 15 minutes is adequate. Stop reaction by washing first in 0.1 M PO4, then in dH2O.

NOTE: The color of the end-product of the DAB reaction may be altered by adding a heavy metal such as nickel or cobalt to the staining solution. Replace PO4 buffer with 0.1 M Tris buffer, pH 7.4.

14. Dehydrate sections through an alcohol series (50%, 70%, 95% twice, absolute alcohol twice, 3 minutes each). Clear in xylene twice, 5 minutes each, and mount in Permount.