Donor Tissue Kindly Provided by Dr. Gloria Pryhuber from the University of Rochester Medical Center

1. For Frozen tissue, rinsed slides for 2X in PBS, then Incubated slides for 5 min in 4% PFA/PBS, then rinsed slides 1X in PBS.
2. Rinsed slides for 3 minutes in running dH₂O.
3. Slides were then incubated in 3% Glacial Acetic Acid (S101, Poly Scientific) for 3 minutes.
4. Slides were then incubated in filtered Alcian Blue (S111A, Poly Scientific) for 30 minutes.
5. Wash slides under cold running tap water for 10 minutes.
6. Incubate for 2 minutes in Nuclear Fast Red (S248, Poly Scientific).
7. Wash under flowing dH₂O until excess Nuclear Fast Red is washed away.
8. Incubated slides in 90% ETOH for 20 dips, repeat in different 90% ETOH.
9. Incubated slides in 100% ETOH for 20 dips, repeat in different 100% ETOH.
10. Washed slides in 2X in Xylene, followed by 6 more washes in xylene.
11. Coverslip with Permount. (Permount Toulene Solution UN 1294, Cat # SP-15-500 Fisher Scientific).

Tissue Used:

LMH-D008-RLL-7B3.13

Gender: Female

Age: 20 Months