Note: Gastric tissue was prepared by histological technicians at McMaster University for safety and training reasons. Tissue was cut 3-4 microns thick, and then placed on the slides. The slides were paraffinized to ensure that they would not be dehydrated. I then deparaffinized them so that they could be properly stained. I carried out the following procedure.

1. 24 slides were deparaffinized in 2 changes of xylene for 5 minutes each.

2. The slides were then re-hydrated in a series of graded alcohol. First, they were rinsed in Xylene 3 times for 5 minutes each.

3. They were then rinsed in absolute ethanol 3 times, 1 minute each time.

4. The slides were rinsed in a quenching solution once, for 1 minute.

5. The slides were placed in the absolute ethanol once, for 1 minute.

6. The slides were rinsed in the 95% ethanol for 1 minute.

7. The slides were washed in 70% ethanol for 1 minute.

8. Running water was used for another rinse for 1 minute, after which the slides were ready for ready for staining.

9. Peroxidase quenching solution was made by adding one part 30% H2O2 (Urea Hydrogen Peroxide) to 9 parts absolute methanol. The slides were submerged in quenching solution.

10. Slides were incubated in a moist chamber at 37 C for 10 minutes, and then rinsed with TBS 3 times for 2 minutes.

11. Reagent 1A was added to 3 drops of Reagent 1B. 2 drops of this mixture was added to each section. Slides were incubated in the moist chamber at 37 C for 10 minutes, and then rinsed in distilled water 2 times for 3 minutes.

12. 2 drops of a denaturing solution called Reagent 2 was then added to each section. Slides were then incubated in the moist chamber at 37 C for 20 minutes, and then rinsed with TBS (Tris Buffered Saline) 3 times for 2 minutes.

13. 100 l of Reagent 3, which is a blocking solution, were applied to each section and followed by incubation in the moist chamber at 37 C for 10 minutes. Sections were then drained and blotted, but not rinsed.

14. 100 l of a biotinylated rodent anti-BrdU called Reagent 4 was added to each section. The slides were incubated in a moist chamber at 37 C for 40 minutes. Afterwards they were rinsed with TBS 3 times for 2 minutes.

15. A streptavidin-peroxidase called Reagent 5 was added in applications of 100 l to each section. Slides were incubated in a moist chamber at 37 C for 10 minutes. They were then rinsed with TBS 3 times for 2 minutes.

16. A dab mixture was created by adding 1 drop of reagents 6A, 6B, and 6c to 1 ml distilled water. 2 drops of dab mixture was added to each section and incubated in the moist chamber at 37 C for 2 minutes.

17. 2 drops of Hematoxylin/Reagent 7 was used to counterstain the slides. Slides were then rinsed in tap water.

18. Slides were then put into TBS until the sections turned blue (approximately 30 seconds) and rinsed in distilled water.

19. The slides were dehydrated in the graded series of alcohol, backwards. Running water rinsed the slides for 1 minute.

20. The slides were washed in 70% ethanol for 1 minute.

21. The slides were rinsed in the 95% ethanol for 1 minute.

22. The slides were placed in the absolute ethanol once, for 1 minute.

23. The slides were rinsed in the quenching solution once, for 1 minute.

24. The slides were rinsed in a quenching solution once, for 1 minute

25. They were then rinsed in absolute ethanol 3 times, 1 minute each time

26. They were rinsed in Xylene 3 times for 5 minutes each

27. 2 drops of a mounting fixative called Reagent 8 was applied to the slide and coverslipped.