This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.


Staining Sections with Antibodies Using PAP

(Source: R. BreMiller)

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 min.

3. Remove Triton solution if used and replace with 2% normal goat serum in PBS/BSA/DMSO for 20 min. 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 4C. 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 min each.

6. Treat with secondary antibody diluted according to manufacturers recommendation with PBS/BSA/DMSO for 1 hr at RT.

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 hr at RT.

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 hr 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 to 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 min 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 min each). Clear in xylene twice, 5 min each, and mount in Permount.

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