[Histonet] Hydrating Frozen Tissues embedded in OCT for Sectioning

[Kasai, Miki (NIH/NCI) [E]]

We are trouble-shooting cryosectioning mouse lung tissue.  Often the lung
section tears or breaks apart during sectioning.  In the past, if the lung
section is proving difficult to section, we take the OCT-embedded tissue and
re-embed it back into OCT (basically put fresh OCT into the original mold
and then place the OCT block with the tissue back into the mold such that
the exposed tissue is covered back with OCT).  This is then placed back in
our -80°C.  When sectioning the next day, the tissue is often easier to
section.

One person in our lab tried to resolve the problem by brushing a little bit
of sterile water onto the tissue when sectioning.  This appeared to hydrate
the tissue and it sectioned better.  However, we weren't sure if this was a
good idea or not.  Any feedback would be greatly appreciated.

For background purposes our lung tissue are processed several ways:

1.  Lungs are perfused with PBS, tissue extracted from mouse, placed in
PFA/sucrose for several hours and then embedded in OCT.

2.  Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1), placed
in PFA/sucrose for several hours and then embedded in OCT.

3.  Lungs are perfused with PBS, inflated gently with OCT/PBS (3:1),
embedded in OCT and frozen by immersion into liquid nitrogen (just the
bottom half of the mold is lowered into LN).

Much appreciation,
Miki Kasai
Biologist
Pediatric Oncology Branch
NCI, NIH
CRC, 1W Rm. 1-3-888
10 Center Drive
Bethesda, MD  20892
(301) 496-2318