[Histonet] Thanks on the cryopreservative question.

[Ben]

  Thanks to all that have responded.  You have been a great help.  I'm going
to infiltrate the block with 30% sucrose without the ethylene glycol then
store the slices in the ethylene glycol cryopreservative at -20C.  Looks
like the best course of action.

 

Ben

 

Here is the thread of messages on ethylene glycol cryoprotectant:

 

 

Hello All,

 

I am attempting to slice sheep brainstem and hypothalamus on a cryostat at

-20 C.  The tissue was treated as such:

 

Perfusion through carotid artery (6L of 4% paraformaldehyde)

Postfixation for 24 hours at 4 C

Cryoprotected in a 20% sucrose solution for 5 days at 4 C

Cryoprotected in a 30% sucrose 30% ethylene glycol solution and stored at

-20 C.

 

I am having some major problems with slicing this tissue.  It appears to

never freeze.  Does anyone know at what temperature I should be slicing?

Any opinions on use of a vibratome instead of a cryostat?  I'm trying to

section tissue at 30-40 um.   Thanks in advance for your help.

 

Ben Renquist

UCDavis

 

Response 1:

 

Ethylene Glycol is anti-freeze.   

 

Pam Marcum

 

Response 2:

 

This can be answer.

 

The 30% ethylene glycol solution is your problem.

Next time do it without ethylene glycol.

20% sucrose following by 30% sucrose is good enough to protect the tissue.

Good luck,

 

 

Margaryan

 

Response 3:

 

Ethylene glycol is anti-freeze - don't use it. Just let brain cryoprotect in
30% sucrose - we never bother with a sucrose gradient anymore but you can
use it if you want.

 

You should snap freeze the brain properly and then get good frozen sections
at that thickness at approx -17C.  but if you have anti-freeze in tissue,
you are probably cutting mush at that temperature.

 

Response 4:

 

Ben,

 

The only time I have seen or used the ethylene glycol in sucrose as a
cryoprotectant has been on brains that have been cut on a sliding microtome
(using dry ice to freeze the tissue), for thick sections (30 microns +), for
floating sections.  The sections collapse on the knife, are picked up with a
paint brush, and flattened out in PBS in the well plate for staining before
mounting onto the slide.

 

James Watson HT, ASCP

Facilities Manager of Histology

GNF, Genomics Institute of the Novartis Research Foundation

 

Response 5:

 

I also had some problems sectioning rat brain in the cryostat after
perfusion. I got a lot of folding and curling (always 20 micron). This may
be due to the temperature in the cryostat. I sectioned the fixed tissue at
the same temperature we section fresh tissue. We section fresh tissue at
knife = -13, specimen = -8 with one cryostat and knife = -15, specimen = -11
with our other cryostat. The optimal cutting temperature always varies from
one cryostat to another, it is important to play around with the temperature
if you are getting folding or cracking.. 

Gayle, do you use -17C for specimen and knife (chamber) temperature?

 

Thanks,

Kelly

 

Question to Responders 2 and 3:

 

Thank you for your response.  I already have some tissue that I would like
to stain.  Is it possible to pull out the ethylene glycol by putting into
30% sucrose and changing that out every few days to maintain the ethylene
glycol gradient.

 

Ben

 

Response 6:

 

You can try. I am not sure. Ask our folks.

Good luck

 

Response 7:

 

I would just immerse in 30% sucrose, it will exchange without a gradient,
just get rid of the ethylene glycol as fast as possible.

 

This whole storage of sucrose cryoprotected tissue problem was just
discussed.  You can store in  30% sucrose at 4C until you need to snap
freeze and avoid storing at -20C. I don't recall people using ethylene
glycol at all.  the tissue IS fixed, and I don't think anything grow in 30%
sucrose  Or snap freeze your samples after 30% sucrose cryoprotection, and
store frozen blocks at -80C, this is what we do, until you need to
equilibrate to cutting temperatures.

 

I would not want even a trace of ethylene glycol contamination around any of
my tissues -  I avoid alcohol fixatives for this reason too.

 

Fiddling with using EG gradient, then undoing it is a pain and a lot of 

unnecessary work plus it is messing up your frozen sections.    I do not 

recall ever seeing EG used for this purpose - is this something you have as
a protocol?  if so, from where?

 

Gayle Callis

 

Question to Responder 6:

 

Thanks, one more question, then I'll quit bothering you.  Is there any
chance that a vibratome might work on this tissue or would I have similar
problems with that?

 

Ben

 

Response 8:

 

I think it can, I usually do agarose embedded tissue for vibratome, but I
think it can also work. Please, let me know when you will finish: if it
worked?

 

All best,

Naira