Re: [Histonet] Mouse lung fixation
In a message dated 1/9/2005 5:44:15 AM US Mountain Standard Time,
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
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
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,
Good luck, hope this helps!
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