616.1 URMC HTC BSL2+ Formalin-Inflated, Paraffin-Embedded Human Lung Tissue

Record details of procedure in Worksheet or Directly in Inventory or ELN

Keep photographic recording of inflation and blocking of lung lobe and tissue

Inflation fixation procedure should be accomplished in a biosafety cabinet with the operator taking appropriate blood and body fluid, including COVID-19 precautions.

Keep the surface of the lobe / tissue moist at all times

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.

Fill the syringe barrel with 10% Neutral Buffered Formalin (NBF)

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

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.

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.

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.

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.

Document volume of fixative infused and comment on any leakage (with degree -minimal, moderate, severe)

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.

Photograph anterior and posterior sides before and after inflation.

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.

Place in the cold room for 40-48 hours

Once tissue has been in fixative for 40-48 hours, processing may continue on grossing station or fume hood

Forty to 48 hours after fixation begins, divide the lobe as diagramed in attached figures into approximately 0.5 cm thick, 1.0 x 1.0 cm² cubes, placing each cube in an appropriately labeled tissue cassette

Place back in 10% formalin in the cold for 18-24 hours.

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.

Tissues may be stored in 70% EtOH in cold until further processed into paraffin.

Remove excess tissues around trachea and large airways at hilum

Collect lymph nodes, excess large vessels, esophagus, spleen and nerves (Best to place these back in shipping buffer and Tend to Lung First)

Weigh each tissue collected

The non-lung samples are sectioned into approximately 0.5-1 cm3 portions

Divide these tissues between 10 % Formalin and PFAS-Cryoprotection

Record these tissues and how processed in BRINDL database

For 10% formalin fixation, place in labeled tissue cassette in formalin; stored at 4⁰C to fix for 64-72 hours (consistent with lung tissue fixation time). Volume of fixative to tissue should be approximately 10:1.

64-72 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

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 rotocol)

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

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` 

Remove cassettes out of processor to embed

Place cassettes in holding tank in the embedding center until paraffin is melted (~15 minutes)

Embed tissue in metal mold as it was in the processing cassette

Cool and solidify blocks on cold plate on embedding center

When blocks “pop” remove them from molds.

Clean outside of cassettes of excess wax and trim excess wax from blocks with the paratrimmer

Store blocks at room temperature away from heat and sunlight

Cool blocks on ice

Using microtome, trim into block at a higher thickness (~14 microns) until a full section is achieved.

Cool block on ice

Section blocks at 4-5 microns into a ribbon

Place ribbon into cool water bath

Place section on slide

Place section into hot water bath

Melt section onto slide

Dry slide standing up in rack overnight or until dry

Store slides in slide boxes at room temperature away from heat and sunlight