[Drosophila] cryosections for laser capture microscopy

[Kiyoteru Tokuyasu]

Dear Dr. Anna Hitrik,

Dan forwarded your mail to me. Let me describe my opinion on your  
problem.

First, I would like to point out that components of O.C.T. are  
polymers, which means that it may fill in intercellular spaces but not  
penetrate into the cell interiors through intact cell membrane.   
Hence, when the ovary is frozen, ice crystals will be formed inside  
the cells, damaging the cellular structures, and as the sections are  
thawed, artificial spaces will be formed in the cytoplasm.

Secondly, as you know, cell structures are more difficult to recognize  
in thicker sections than in thin ones, mainly due to super-positions.   
This will be particularly true in unstained sections.

If cryosections thinner than 7 micron thickness are OK or preferable,  
there is a way to obtain 0.1-2 micron thick cryosections rather  
readily. The cutting quality of thin cryosections is much improved by  
infusing a sucrose solution into cells (Tokuyasu, K.T. 1973. A  
technique for ultracryotomy of cell suspensions and tissues. J. Cell  
Biol. 57:551-565).
    A formaldehyde-fixed Drosophila ovary may be infused with 1.5-2.3M  
sucrose in an appropriate buffer, frozen by plunging the specimen into  
liquid nitrogen and sectioned at -70~ -90oC.   If your cryo-microtome  
is not suitable for cutting such thin cryosections, you may try to  
find a nearby EM lab equipped with an ultra-cryomicrotome and carrying  
out immunostaining of ultrathin cryosections.   Such lab will know how  
to retrieve cryosections onto glass slides and remove sucrose from the  
sections.

You may then have a fair chance to see cell boundaries in thin, thawed  
cryosections, with phase-contrast LM or other means.

If your project requires to see much finer structural details, I would  
propose a new approach;  all operations involving the laser capture  
microscopy are done first to obtain a data “map” and then the  
cryosection is stained to obtain a morphological “map”.  Using an  
appropriate coordinate, one may combine the two “maps” to obtain the  
whole “map”.  Such an approach could also be used for your 7 micron  
thick section, if necessary.

I would hope that these suggestions were useful to you.

Sincerely,

K. Tokuyasu