1. Set up and document date of harem breeding.
  2. Day mice are born is recorded as PND 0.
  3. Record date harvested.
  4. Set up the inflation fixation apparatus. The apparatus consists of a ring stand, a clamp, and a 3cc or 10cc syringe barrel. Attach a 23 Gauge butterfly to the syringe barrel with a clamp to be able to stop the flow of PFA through the butterfly. Put the syringe barrel through the ring stand clamp. Fill the syringe barrel with 4% PFA/PBS prepared with DEPC treated water and allow to flow completely through the butterfly. Clamp the butterfly to prevent the flow of 4% PFA/PBS and add 4% PFA to the barrel of the syringe to fill the syringe barrel. Adjust the barrel so that the meniscus of the 4% PFA solution is 25 cm above the trachea of the mouse.
  5. Inject mice with 0.1ml pentobarbital. Wait until mouse is unresponsive by toe pinch.
  6. Spray the abdominal surface with 70% ETOH.
  7. Isolate a small piece of tail for isolating genomic DNA to determine gender if unable to visually determine gender.
  8. Make a small cut with scissors at the pubis followed by a longitudinal cut along the central midline through to the chin.
  9. Dissect away the skin leaving the abdominal wall intact.
  10. Open the abdominal cavity by grasping a small piece of muscle and making an incision.
  11. Exsanguinate mouse by cutting the inferior vena cava
  12. Insert one blade of scissor under the rib cage and cut through the midline while applying upward pressure on the bottom scissor blade.
  13. Dissect away the diaphragm.
  14. Separate the rib cage and remove as much of the rib cage as possible to expose the heart and lungs.
  15. Dissect away the salivary gland with forceps.
  16. Dissect away the muscle around the trachea to fully expose the trachea.
  17. Tie a knot around the trachea with a suture but do not fully close the knot around the trachea.
  18. Make a small incision in the ventral side of the trachea with a pair of scissors and insert the butterfly bevel side up. Carefully close the suture around the trachea and inserted needle. Alternatively, insert the cannula through the mouth/larynx.
  19. Open up the butterfly clamp allowing the 4% PFA/PBS to flow into the lung. Once the lungs are inflated to the correct level of inflation fixation, clamp the butterfly to prevent the flow of 4% PFA/PBS.
  20. Remove the butterfly and completely close the suture knot around the trachea.
  21. Remove the lung, heart and thymus en bloc.
  22. Place the dissected tissue in a 50ml conical tube containing 20ml 4% PFA/PBS.
  23. Place on ice and then place at 4oC overnight.
  24. Following day wash 3X in 1X PBS made with DEPC treated water for 10 minutes each wash at room temperature.
  25. Dissect away the individual lung lobes (Right Upper, Right Middle, Right Lower, Left Lobe, and accessory lobe). Lung lobes (right upper, right lower, and the left lobe) were bisected near the large bronchi with a razor blade. To maximize tissue for frozen and paraffin embedded tissue,  The upper portion of each bisected upper right, lower right, and left lobes and the intact right middle lobe were processed for paraffin embedding as described in step 30. The lower portion of each bisected upper right, lower right, and left lobes and the intact accessory  lobe were processed for OCT embedding as described in step 26.
  26. Place lungs in 30% Sucrose/1X PBS (DEPC treated water).
  27. Incubate lungs O/N in 30% Sucrose @ 4o Alternatively, tissue can be stored in this solution @ 4oC.
  28. Day before freezing place lungs in 2 parts 30% Sucrose and 1 Part OCT and incubate @ 4o
  29. Alternatively, lungs can be prepared for paraffin embedding.
  30. Following step 25, lungs are dehydrated through 30% ETOH, 50% ETOH, and 70% ETOH washes 3X for 10 min each wash. Lungs can then be stored in 70% ETOH at 4oC until processed on tissue processor and then embedded in paraffin. The bisected lobes were embedded with the cut side facing down into wells. (This maximizes the viewing of proximal and distal regions of the lung and extends the amount of tissue available).