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Murine lung fixation - OCT alternatives? (Jan/20/2005 )

Hi,

I am looking to stain murine lungs with various immune cell antibodies and have been told that more antibodies are available if the tissue is cryo frozen vs. formalin fixed. However OCT compound is too thick to get into the lung via the trachea and does not seem to permeate the lung well from the outside. Anyone have any other ides?? huh.gif

Thanks

-imperial-

It depends on what cells you are looking for. Yes more antibodies are available for frozens than paraffin embedded but you can get the pertinent antibodies for paraffin.

I only use paraffin sections as the morphology is superior to frozens. I have a panel of about 20 antibodies to various immune cells and all work with paraffin. It may take a bit longer but you should be able to hunt down what you want and have it compatible for paraffin.

-scientist-

Hi,

If you want to "inflate" the lungs via trachea before isolating them, you can use a 1:1 dilution of OCT with sterile PBS. This will solve your viscosity problem. After this, you'll have to imerse the isolated lung into OCT compound before freezing. However, I found cryosections from "noninflated" lungs to be as good for immunofluorescence staining.

I hope this helps,
Diana

-diana-

I agree with Diana, using the 50% OCT/PBS can work or a 50% OCT/sucrose. I have also done low melting point agarose and it works extremely well - easy to cut too. Also, like Diana, I also found cryosections from non-inflated lungs to stain just fine, the only thing is morphology is extremely compromised. So it depends on your ultimate objective do you want speed, staining quality, and/or morphology.

It is true that many immune cell Abs will not work on paraffin or even PFA fixed frozens in my experience. Or perhaps they might work sometimes and not other times so reproducibility can be low. I always prep both a paraffin and a frozen block for this purpose. However a lot does depend on antibody source and the particular person doing the stain so I would try a few out on various preparations before you commit to a large scale study.

Good luck.

-MaximinaNYC-

In regards to this can anyone give a more detailed protocol for inflation fixing of the lung?
Or could Diana or others who have had good luck with "noninflation" methods provide a protocol for that?
The more idiot-proof the better! rolleyes.gif
Thanks!

Jess

-Jessgene-