Congo Red (CR) Staining – Fat Pad Biopsies

1. Label slides with patient name.
2. Place specimen from syringe on slide and place second labeled slide on top (at 90° angle and   face to face with bottom slide).
3. Squeeze two slides together firmly with thumb and index finger.
4. Remove top slide up and off bottom slide (DO NOT REMOVE AS FOR A BLOOD SMEAR. This is a squash not a smear.).
5. Air dry slides for 10 min. (fat sample never really dries in this time, but adheres better if you do this)
6. Use a CR positive control slide.
7. Prepare in staining dishes fresh 200 mlsolutions of:
   • 10% buffered formalin
   • ethyl alcohols (95%, 100%) and xylene
   • Mayer’s Hematoxyline
   • alkaline 80% ETOH/saturated NaCl: add 2 ml of 1% NaOH to 198 ml 80% ETOH/saturated NaCl and filter. (Use within 15 mins.)
   • alkaline CR: add 2 ml of 1% NaOH  to 198 ml of stock CR solution and filter. (Use within 15 mins.)
8. Place all slides in staining rack.
9. Fix slides in formalin (under hood) for at least 10 min.
10. Wash gently 3x with ddH2O.
11. Put in 2 separate 95% alcohol solutions (10 dips in each until slides are completely bathed), blot bottom of rack with paper towel to remove excess solution before proceeding to the next rinse).
12. Wash gently with ddH2O.
13. Place in Mayer’s hematoxylin for 1 min.
14. Place slides at one end of large slide dish and run tap water on opposite end of dish for 2 min.
15. Wash gently 3x with ddH2O.
16. Place in alkaline 80% ETOH/saturated NaCl for 20 min.
17. Stain in alkaline CR for 20 min (dip intermittently).
18. Place in 95%, 95%, 100%, 100% alcohol solutions respectively followed by two separate xylene rinses (dip repeatedly until slides are completely bathed, blot bottom of rack with paper towel to remove excess solution before proceeding to the next rinse).
19. Place coverslips on slides using permount solution.
20. Clean off excess permount by dipping in xylene solution.
21. Apply sticky label with date, sample number, patient name and “Congo Red”.