I'm looking for tips on how to freeze mouse lungs in OCT. My currently I remove the heart-lung block and insert a cannula in the trachea, then inflate with 600-800 ul of OCT:PBS 1:1. I let it sit in this mixture until I am able to freeze it (often a few hours later because I need to move to another building). When I am ready to freeze I gross the lung into the separate lobes and place them in a little OCT on the bottom of a mold. Then I cover with OCT and set it in cold 2-methylbutane over liquid nitrogen until frozen.

My lungs looked sort of half-inflated under the microscope. I am wondering if I need to tie off the trachea after inflating. I am also finding that when I cut the lobes apart I get some air leaking out, which makes it hard to avoid bubbles in the OCT. I think my lungs are trying to float in the OCT sometimes as well. I am hoping to avoid fixative as I need to use an antibody that I know doesn't work in fixed, paraffin embedded tissue.

Would appreciate any advice you can give!