Donor tissue kindly provided by Dr. Scott Randell, University of North Carolina

1. For Frozen tissue, rinse 2X in PBS, then 5 min in 4% PFA/PBS, then rinse 1X in PBS.
2. Rinse slides 2X in dH₂O for 20 dips each rinse.
3. Rinsed slides for 3 minutes in running dH₂O.
4. Slides were then stained in filtered Hematoxylin for 60 seconds. (Gold Standard Harris Hemotoxylin, Acidified, Mercury; Cat # 24245 Polysciences Inc,).
5. Rinsed slides in tap water until water turned cleared then rinsed in dH₂O
6. Rinsed slides in Lithium Carbonate: 20 dips. (Lithium Carbonate ACS Grade; Cat # 255823-500G Sigma Aldrich). Lithium carbonate is prepared by over-saturating in dH₂O.
7. Rinsed slides in dH₂O 3X 20 dips.
8. Rinsed slides in 70% ETOH for 20 dips.
9. Rinsed slides in 80% ETOH for 20 dips.
10. Stained slides in Eosin for 15 seconds. (Eosin Y 0.5% Alcohol Solution, Cat # 09859 Polysciences Inc,).
11. Rinsed slides in a series of 95% ETOH washes. 10 dips for the first and second washes, 15 dips in the third and fourth washes.
12. Rinsed slides 3X in 100% ETOH. 10 dips for the first and second washes and 15 dips in the third and fourth washes.
13. Rinsed slides in 2X in Xylene, followed by 6 more washes in xylene.
14. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMHA-15-UNC-DD036L-04HF-1-1, 17, 35

Gender: Male

Age: 47 Year Old

Race: Caucasian

Non-Smoker