[Histonet] Mouse lung fixation

[RCHIOVETTI <@t> aol.com]

In a message dated 1/9/2005 5:44:15 AM US Mountain Standard Time, 
Andrew.MacDuff <@t> ed.ac.uk writes:
am just starting a research project and need to fix mouse lungs for embedding 
in parafin so that I can use them for histology and immunohistochemistry. 
Could you please tell me the best way to fix the lungs? So far I have tried 
removing the lungs and then instilling 10% neutral buffered formalin into the 
trachea with a syringe. This seems to work but I'm not sure how reproducible the 
alveolar architecture will be. Also do people exsanguinate the mouse before 
fixing the tissues? (we've been using cervical dislocation and then just removing 
the lungs)
Andrew,

I haven't done lung tissue for paraffin embedding, but I've done a lot of 
mouse lung prep for EM (using glutaraldehyde instead of formalin).  

I would recommend perfusion fixation.  It's good enough for EM, so it should 
work great for light microscopy.

Contact me off-list if you need additional details.  In a nutshell, our 
protocols usually went something like this:

1.  We used chemical anesthesia via intraperitoneal injection, but cervical 
dislocation should be OK as long as you can get into the chest cavity while the 
heart is still beating.

2.  Use a 26 ga. needle and an IV tubing set with a "piggyback" port on it.  
I like the small butterfly needles like the ones that are used for pediatric 
scalp veins...gives you something to hold onto when you go in with the needle.

3.  Pre-fix washing solution: Ringer's saline + 0.5% procaine + saccharose to 
adjust osmolarity to about 350 mOsm (the procaine relaxes the circulatory 
system's musculature and ensures good access to the lung's capillary bed and the 
alveoli).  Warm to 37 degrees C and hang in an IV bag about 1 Meter above the 
animal.

4.  Remove the sternum and the anterior portions of the ribs in one piece to 
expose the heart.

5.  Move quickly from this point on.  Insert the needle through the heart 
wall into the right ventricle.

6.  Start the flow of the wash solution and at the same time make a nick in 
the left atrium.  This gives you a direct route to the pulmonary circulation.

7.  Perfuse for about 1 to 1-1/2 minutes.  You will know everything's working 
OK if you see the lungs blanch from pink to white.

8.  "piggyback" the fixative solution (NBF?), also warmed to 37 degrees and 
hanging in another IV bag / tubing setup, into the line that's in the heart.  
Shut off the washing buffer and start the fixative flowing.

9.  Perfusion fix for about 10 minutes in-situ.  After that you can remove 
the lungs and dissect them in more fixative.

10.  For EM studies the fixative solution was also adjusted to 380 mOsm with 
saccharose.  I don't know whether this is absolutely necessary for histology, 
though.

Good luck, hope this helps!

Cheers,

Bob Chiovetti