PGP 9.5 (Ubiquitin terminal carboxylase) Immunodetection Procedure

1. Begin with sectioned tissue on gelatin-subbed or permafrost slides.

2. Wash 3x10 minutes in 0.1 M Phosphate buffer (PB)

3. Incubate 5 minutes in 0.1% H₂O₂ in PB. (1/30 of 3% H₂O₂)

4. Wash 2x10 minutes in PB.

5. 1-2 hourS in 10% donkey serum in 0.1 M PB + 0.3% Triton X-100 + 2mg/ml BSA.

for 5 mL of antiserum media:

500 µL serum, preferably from the secondary host species
2.5 mL 0.2 M PB
500 µL 3% Triton
1.5 mL dH₂O
10 mg Bovine Serum Albumin

6. Overnight at 4° C in primary antibody (rabbit anti-PGP) diluted 1:1000 in antisera media. Each slide needs ~200 µL to be covered. Slides should be kept in sealed humid chamber to prevent drying,coverslips made with Parafilm can be used to prevent drying out.

7. Wash 3X10 minutes in 0.1 M PB + .1% Tween.

8. Overnight at 4° C or 2 hours at room temperature in secondary antibody (biotinylated donkey anti-rabbit) diluted 1:500 in antisera media. Use same technique as in step 6.

9. Following incubation, prepare ABC solution by adding 9µL each of the A and B solutions in antisera media without the normal serum ABC should sit at room temperature for ~30 minutes before using.

10. Wash slides 3x10 minutes in 0.1 M PB + 0.1 % Tween.

11. Incubate 2 hours at room temperature in enough ABC to cover each slide. Again, used sealed humid chamber to prevent dying.

12. Wash 3x10 minutes in 0.1 M PB + 0.1% Tween.

13. Perform DAB reaction:

dissolve one tube DAB in 50 mL 0.1 M PB + 0.1% Tween-20.
incubate slides in DAB solution 10 minutes.
- add 60 µL 3% H₂O₂ per 50 mL DAB solution and incubate 5-15 minutes until sufficient color develops.

14. Wash 2x5 minutes in 0.1 M PB.

15. Slides can be dehydrated through ethanols to xylene and coverslipped in Permount.