Hi Angelo

I've instilled fluid many times into trachea + lungs; it's not an issue to have deflation when you take the trachea + lungs out, they will reinflate upon instillation (ie. it's not "irreversible"). I've not done it with OCT but I'm told it's commonly done; not sure just a 1:1 dilution in PBS would be enough as OCT is pretty thick, but you can try). I wouldn't try to do it in situ myself, as I'm not sure you'd be able to flush all the air out while instillation is occurring, you might have trapped air which could cause issues with freezing.

You would inflate the lungs with your OCT solution at room temperature, immediately place them in the mold, fill the mold with OCT (RT) to surround and cover the sample (avoiding bubbles), and then freeze immediately. Your mold should be the correct size so that you have to put in as little OCT as possible, since excess OCT could slow down the freezing, as you mentioned. But the OCT should cover the top of the sample.

The isopentane has to be at the correct temperature, so has to be placed into the LN2 a little bit before freezing. The usual criteria is that the internal surface of the metal beaker containing the isopentane should have a fine layer of frozen isopentane (white). Once that layer is there you're ready to freeze. You immerse the whole mold into the isopentane, until the OCT has entirely turned white (usually 30-60 seconds, but for a large sample like this is might take longer). If you're doing a lot of samples at once the frozen isopentane may start to fill the beaker and make it impossible to submerge your mold, so you may need to pull it out until it's thawed and start over. It helps to have a set up where your beaker is mounted on a stand with clips that allow lowering it into the LN2 and lifting it out easily. Of course you have to be careful with the LN2. Isopentane is also flammable so you have to be careful to avoid open flames or sparks in the area. Not sure about n-heptane, I haven't used that.

Hi Jean-Martin

Thanks a lot for your answer.

Yes, I have read about the process of cooling isopentane into LN2. I am actually going to use it in order to cryopreserve skeletal muscle. However, according to some papers, cryopreservation of lungs with n-heptane gives better results compared with isopentane and this is why I was thinking to use the former.

However, I truly prefer to use isopentane as n-heptane seems also to be toxic and thus I would like to avoid it. Have you been satisfied so far with the quality of your freezing in the lungs when you use isopentane? Is the alveolar structure of your specimen well preserved? If you have done any studies where you have analysed alveolar structure (i.e wall thickness, number etc), it would be wonderful in case you could share the manuscript.

Thanks.

Angelo

I've not done much examination of frozen lung sections. The large majority of our work is done on FFPE tissue, we only work on frozen tissue in the rare cases where we're looking at IHC/IF for markers that don't work on FFPE. If you're looking for good morphology preservation, you might be disappointed with frozen sections, which are much more difficult to do and require a fair bit of training to do well, and even then are often prone to artifacts.