TITLE: 611.2 URMC HTC Formalin-Inflated, Paraffin-Embedded Human Lung Tissue
AUTHORS: Gloria S Pryhuber, Heidie Huyck, Daria Krenitsky

[DESCRIPTION]
Processing of Formalin Fixed Lung and Non-Lung Tissue for the LungMAP HTC 


[STEPS]
SECTION: Record Process
1. Record details of procedure in Worksheet or Directly in Inventory or ELN
SECTION: Record Process
2. Keep photographic recording of inflation and blocking of lung lobe and tissue
SECTION: Inflation Fixed (Formalin) Lung Lobe
3. Inflation fixation procedure should be accomplished in a grossing station or biological safety cabinet with the operator taking appropriate blood and body fluid precautions.
SECTION: Inflation Fixed (Formalin) Lung Lobe
4. Keep the surface of the lobe / tissue moist at all times
SECTION: Inflation Fixed (Formalin) Lung Lobe
8. Cannulate the main bronchus, identified by its cartilage containing wall, with an endotracheal tube or 16 or 18 gauge catheter. 

Fix the airway tube in place with suture material or zip tie. 

If two bronchi are apparent due to a branch-point, each can be cannulated. 

Tying in of the airway tubes is the “trickiest” part of the entire procedure and is most easily done by two people.
SECTION: Inflation Fixed (Formalin) Lung Lobe
9. Connect the IV extension tubing coming from the syringe to the airway tube(s). 

If more than one bronchi is cannulated, they can be instilled serially or simultaneously with a second syringe set up. 
SECTION: Inflation Fixed (Formalin) Lung Lobe
10. Release the clamp and allow formalin to infuse into the lung lobe that should visably inflate, the degree of inflation varies.

 Transient, gentle pressure on the fluid head may be needed to start the flow of fixative.
SECTION: Inflation Fixed (Formalin) Lung Lobe
11. Add NBF to the syringe to maintain meniscus at 25 cm water inflation pressure. Keep track of volume added.

Maintain instillation pressure for approximately 10 minutes after fluid stops flowing.
SECTION: Inflation Fixed (Formalin) Lung Lobe
5. Connect a short piece of IV extension tubing, clamped with hemostat or shut off with stopcock, to a 10 or 20 ml syringe barrel (without plunger) suspended in a clamp on a ring stand so that the 5 ml syringe mark is 25 cm above the level of the main airway in the lobe to be fixed.
SECTION: Inflation Fixed (Formalin) Lung Lobe
6. Fill the syringe barrel with 10% Neutral Buffered Formalin (NBF)
SECTION: Inflation Fixed (Formalin) Lung Lobe
7. Flush the IV extension tubing through with formalin to remove air bubbles and so the NBF fluid level is at 10 or 20 ml mark on the syringe. Reclamp. prior to connection to the instillation cannulas
SECTION: Inflation Fixed (Formalin) Lung Lobe
12. Document volume of fixative infused and comment on any leakage (with degree -minimal, moderate, severe) 

SECTION: Inflation Fixed (Formalin) Lung Lobe
13. Photograph anterior and posterior sides before and after inflation. 
SECTION: Inflation Fixed (Formalin) Lung Lobe
14. Once instillation is complete, pull the suture or zip tie that is on the airway and airway cannula tight while removing the cannula. 

This is best done with a second pair of hands. Also helpful to have a hemostat available as an emergency clamp. 
SECTION: Inflation Fixed (Formalin) Lung Lobe
15. Place the securely tied lung lobe in a specimen container filled with 10% NBF.

For good fixation, prefer with a volume ratio of 10:1 fixative:lung.
SECTION: Inflation Fixed (Formalin) Lung Lobe
16. Place in the cold room for 20-24 hours
SECTION: Inflation Fixed (Formalin) Lung Lobe
17. Twenty to 24 hours after fixation begins, divide the lobe as diagramed in attached figures into approximately   thick, 1.0 x 1.0 cm2 cubes, placing each cube in an appropriately labeled tissue cassette

SECTION: Inflation Fixed (Formalin) Lung Lobe
18. Place back in 10% formalin in the cold for 18-24 hours.
SECTION: Inflation Fixed (Formalin) Lung Lobe
19. The tissue cubes are then rinsed in 1xPBS and dehydrated by 20-minute  successive dwell times in PBS, 30% EtOH, 50% EtOH and 70% EtOH made  with diethylpyrocarbonate (DEPC) treated water.
SECTION: Inflation Fixed (Formalin) Lung Lobe
20. Tissues may be stored in 70% EtOH in cold until further processed into paraffin.
SECTION: Formalin Fixed Non-Lung Tissue
21. Remove excess tissues around trachea and large airways at hilum 
SECTION: Formalin Fixed Non-Lung Tissue
22. Collect lymph nodes, excess large vessels, esophagus, spleen and nerves (Best to place these back in shipping buffer and Tend to Lung First) 


SECTION: Formalin Fixed Non-Lung Tissue
22.1. Weigh each tissue collected 


SECTION: Formalin Fixed Non-Lung Tissue
22.2. The non-lung samples are sectioned into approximately 0.5-1 cm3 portions 

SECTION: Formalin Fixed Non-Lung Tissue
23. Divide these tissues between 10 % Formalin and PFAS-Cryoprotection 

Record these tissues and how processed in BRINDL database 
SECTION: Formalin Fixed Non-Lung Tissue
23.1. For 10% formalin fixation, place in labeled tissue cassette in formalin; stored at 40C to fix for 40-48 hours. Volume of fixative to tissue should be approximately 10:1. 

Forty-48 hours after fixation begins, the tissues are rinsed in 1xPBS and dehydrated by 20 minute successive dwell times in PBS, 30% EtOH, 50% EtOH and 70% EtOH made with diethylpyrocarbonate (DEPC) treated water. 

Tissues may be stored in 70% EtOH in cold until further processed in paraffin
SECTION: Formalin Fixed Non-Lung Tissue
23.2. For Freezing in OCT: fix in 4% PFA x 20-24 hrs, cryo-protect in 30% sucrose and continue to process for freezing in OCT along with lung tissue to be frozen (See PFA fixed, sucrose cryoprotection protocol) 
SECTION: Tissue Processor
24. From 70% ethanol, place cassettes into metal basket in VIP processor’s retort (up to 150 unicassettes or 75 megacassettes) in 70% ethanol already in the retort 




SECTION: Tissue Processor
25. Use overnight program #3 on VIP Processor 
    70% Ethanol – 1 hour – 40 °C 
    80% Ethanol – 1 hour – 40 °C
    95% Ethanol – 1 hour – 40 °C 
    95% Ethanol – 1 hour – 40 °C 
    100% Ethanol – 1 hour – 40 °C 
    100% Ethanol – 1.5 hours – 40 °C
    Histological grade Xylene – 1 hour – 40 °C 
    Histological grade Xylene – 1.5 hours – 40 °C
    McCormack’s Paraplast (paraffin) – 1 hour – 60 °C 
    McCormack’s Paraplast (paraffin) – 1 hour – 60 °C 
    McCormack’s Paraplast (paraffin) – 1.5 hours – 60 °C 




SECTION: Tissue Processor
26. Remove cassettes out of processor to embed 




SECTION: Embedding Tissue
27. Place cassettes in holding tank in the embedding center until paraffin is melted (~15 minutes) 




SECTION: Embedding Tissue
28. Embed tissue in metal mold as it was in the processing cassette 




SECTION: Embedding Tissue
29. Cool and solidify blocks on cold plate on embedding center 




SECTION: Embedding Tissue
30. When blocks “pop” remove them from molds. 




SECTION: Embedding Tissue
31. Clean outside of cassettes of excess wax and trim excess wax from blocks with the paratrimmer 




SECTION: Embedding Tissue
32. Store blocks at room temperature away from heat and sunlight 




SECTION: Sectioning Paraffin Embedded Tissue
33. Cool blocks on ice
SECTION: Sectioning Paraffin Embedded Tissue
34. Using microtome, trim into block at a higher thickness (~14 microns) until a full section is  achieved.
SECTION: Sectioning Paraffin Embedded Tissue
35. Cool block on ice
SECTION: Sectioning Paraffin Embedded Tissue
36. Section blocks at 4-5 microns into a ribbon
SECTION: Sectioning Paraffin Embedded Tissue
37. Place ribbon into cool water bath
SECTION: Sectioning Paraffin Embedded Tissue
38. Place section on slide
SECTION: Sectioning Paraffin Embedded Tissue
39. Place section into hot water bath
SECTION: Sectioning Paraffin Embedded Tissue
40. Melt section onto slide
SECTION: Sectioning Paraffin Embedded Tissue
41. Dry slide standing up in rack overnight or until dry
SECTION: Sectioning Paraffin Embedded Tissue
42. Store slides in slide boxes at room temperature away from heat and sunlight