Quorum Sensing

Take hold of the arteries below the lungs using tweezers and

cut the arteries with a pair of scissors. Lift the lungs upright and

cut the arteries next to the heart. The lungs should now be

isolated with the tweezers.

4. Place the isolated lungs in 5 ml of 0.9% (wt/vol) NaCl in a
   15 ml centrifuge tube and keep it on ice until homogenization.


5. Sterilize the surgical equipment between each mouse using
   70% (vol/vol) ethanol.


6. Homogenize the lungs using a homogenizer. The crusher is
   washed in 35 ml 70% (vol/vol) ethanol for 5 s, for 20 s in 35 ml
   70% (vol/vol) ethanol, for 5 s in 35 ml 0.9% (wt/vol) NaCl,
   and for 5 s in 30 ml 0.9% (wt/vol) NaCl. Use 50 ml centrifuge
   tubes for the washing. All the tubes are changed after 5–8
   samples. Between groups the homogenizer must be taken
   apart, cleaned, and sterilized. Please notice that the homoge-
   nizer must only be used when placed in fluid.


7. Make tenfold serial dilutions of each lung homogenate and
   plate all the dilutions onto blue agar plates. Incubate the plates
   at 37C overnight.


8. Count the plates and calculate the CFU/lung (seeNotes 5
   and 6 ).

3.4 Freeze
Microtomy

1. After a chosen period of time (seeNote 7), the mice are
   euthanized using pentobarbital (10.0 ml/kg body weight)
   injected intraperitoneal (i.p.). Ensure that local animal care
   regulations are followed.


2. Fixate each dead mouse on its back and apply 70% (vol/vol)
   ethanol on the chest and stomach to avoid contamination.


3. Isolate the lungs by first removing the skin with a scissor and
   tweezer. Then open the thoracic cavity without damaging the
   lungs. Take hold of the arteries below the lungs using a twee-
   zers and cut the arteries with the scissor. Lift the lungs upright
   and cut the arteries next to the heart. The lungs should now be
   isolated with the tweezers.


4. Whole lungs are placed in the Tissue-Tek mold and covered
   with Tissue-Tek O.C.T.™gel. Hexane is cooled down using
   dry ice (seeNote 8) and the embedded lungs are placed in the
   cooled hexane for 5–10 min. Keep it on dry ice afterwards. This
   should happen as soon as possible after isolation of the lungs.


5. Cool the Tissue-Tek Cryo 2000 to operating temperature and
   make different sections of the lung tissue. Frozen sections
   should be 10–50μM thick.


6. Place the sections on cover glasses for microscopy inspection.


7. Study the lung sections with epifluorescence or CSLM (see
   Note 9).

210 Louise Dahl Hultqvist et al.