[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


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[Histonet] Immunofluorescence staining/minimizing background staining

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

Hi,

We are performing some immunofluorescence staining on mouse lung tissue.  We
are getting some nice positive staining with some of our initial antibodies
(procollagen, cytokeratin).

We would like to minimize the amount of background staining we are getting.
We are titering our primary antibodies to find out optimal Ab concentration
as well as the secondary conjugate Ab with the fluorophore of interest.  We
use donkey serum for general blocking.

Any other suggestions?

Much appreciation,
Miki


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[Histonet] Using Nail Polish on coverslip?

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

We are currently starting up some IHC on frozen tissue sections.  After 
staining with different fluorescent antibodies, we end with applying DAPI 
w/Prolong gold and then coverslipping.  We would like to seal the coverslip so 
that we can keep the slides longer.

Any suggestions on where and how best to apply the nail polish for a permanent 
fix on the coverslips?

Thanks,

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