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 th