[Histonet] Re: human skin frozen sections Attn Shanu

[gayle callis]

Shanu, 

 

You can also fold the skin slightly into a V shape so the hair is on the
INSIDE of the fold.  Hold this V folded tissue in forceps, embed in OCT,
then allow to start freezing, then release the hair, add more OCT to the
unfrozen OCT.  

 

We have frozen bovine (very hairy!) and mouse skin into this configuration,
and after freezing, be sure to equilibrate your block for 20 minutes or so,
to the cutting temperature inside the cryostat.  Be sure you are not taking
a freshly mounted block from a very cold area of cryostat and try to cut it
at -21C unless you have the tissue equilibrated to that cutting temperature.
We have a (beloved!)Cryocut 1850, and the freezing plates where the Peltier
device is located are always 10 degrees colder than the object/blade
temperature.  We set our mounted blocks next to the microtome for
equilibration to the cutting temperature (takes approx 20 minutes).  

 

We have the blade, object and chamber temperature all the same, -20C
although you can try colder or warmer. Equilibration from freezer to
sectioning temperature goes faster if you mount the tissue on the metal
disk, then let it equilibrate next to microtome.  Waiting for things to warm
up, freeze onto disk, then warm up to cutting temperature tries ones
patience. We temperature tested to know where all the warm and cold spots in
the chamber are located, and use the warm areas which are usually the
object/cutting temperature, for block equilibration.     

 

We orient the V shaped tissue so the blade meets the bottom of the v shape
first, and sectioning proceeds through the tissue with the dermis/epidermis
sectioned last. We prefer high profile blades over low profile for more
stability, but just as sharp. We do not increase the blade angle. We have
used Tissue Tek AccuEdge high profile blades, set at 6, one tick past 5, in
our cryostats for over 20 years - they are excellent for frozen sections,
and with skin, change the edge frequently for the sharpest blade. 

 

Gayle M. Callis

HTL(ASCP)HT,MT

Bozeman MT 59715   

 

 

 

You wrote: 

 

I have been sectioning some fresh frozen hairy human skin on the cryostat
and I am having too much trouble getting decent sections. The sections seem
to tear up and the epidermis & the dermis appear to separate.

 

My technique:

I embed dermis side down in OCT compound and then place the mold onto some
dry ice until the mold solidifies. I then transfer the molds to a -80ºC
freezer until they are ready to cut. Next I let the blocks come to cryostat
temperature in the chamber. For cutting, I turn the block at a 90º angle
meaning that both the dermis & the epidermis get cut in the same plane. I
usually cut at a Chamber Temp of -19 & an Object Temp of -21. My cutting
angle is between 3 & 5. I use low profile blades. I trim most of the fat
that may be present in the skin, but there may be residual fat pockets that
I may have missed!

 

This morning I talked to the technical staff at Leica and I was suggested to
change the Chamber Temp to -21 to match that of the Object Temp. Also, I was
asked to try with an angle of 5 or greater than 5 (as high as 8 or 9). I
tried a range of temperatures and cutting angles, all in vain. I am still
not happy with the quality of sections I am getting! 

 

Any suggestions on what I could do? I greatly appreciate any comments.

 

Thanks to all,

Shanu