Overgrown Cell Culture Staining Procedure

1. Remove the Flasks from the incubator and pour off as much medium as possible.

2. Remove the top portion of the flask and discard

3. Quickly drain excess medium off of the slide and place into alcoholic formalin immediately and fix for 30 - 60 min. Do not allow the cells to dry out!

4. Rinse the slide in running distilled for 2 min.

5. Stain in Harris hematoxylin for 15 min.

6. Rinse in running tap water until clear.

7. Blue in 0.25% ammonium hydroxide for 30 sec.

8. Rinse in running tap water for 1 min.

9. Counterstain in 1% Eosin Y w/ 1:10 methanol in 95% ethanol for 5 min.

10. Differentiate in 2 changes of 95% ethanol, 6 dips each.

11. Dehydrate in 2 changes of absolute ethanol, 10 dips each.

12. Remove from alcohol and let air dry.

13. Coverslip using glycerol or other aqueous mounting medium.