[Histonet] Re: Accidental freezing of fish samples

[Sherwood, Margaret]

Gayle,

I would be interested in the articles as well.  I had a similar experience:
nerve samples from rats were being shipped to me in K2 (which I had previously
sent to them).  I specifically said not to freeze the samples; they shipped them
on dry ice!  I was just doing one micron sectioning, but they were useless.  

Thanks!
Peggy  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle callis
Sent: Friday, May 20, 2011 12:27 PM
To: 'Histonet'
Subject: [Histonet] Re: Accidental freezing of fish samples

You wrote: 
 
Probably the bone staining will be OK - how about sending the student to the
arctic circle for a few days in winter? - that may be a good lesson about
not freezing tissues. Just kidding :-)
 
On Fri, May 20, 2011 at 9:24 AM, Tora Bardal <tora.bardal
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> <@t>
bio.ntnu.no>wrote:
 
> One of our students accidentally put his samples in the freezer after
formaldehyde/glutaraldehyde/PBS fixation. The samples (fish very early
stage, of course rare species) were meant for LM/EM morphology studies.
Good morphology is out, but is there a chance > he can do cartilage/bone
staining? Or do we need to send him abroad for new sampling at the next
spawning time?
> Tora Bardal
> Senior Engineer
> NTNU Center of Fisheries and Aquaculture
 

I was amused by Louise's reply.  Your comment "good morphology is out" is
correct, especially for EM where bone and cartilage may NOT be optimal after
a freeze/ thaw.  EM requires optimal fixation and handling to have the best
results.  There will probably be too much freezing artifact damage (large
water ice crystal formation) in the EM samples.   However, if doing light
microscopy and not worried about soft tissues and with paraffin processing ,
the bone and cartilage may be ok, but not entirely be ideal ......individual
bone and cartilage cells, along with soft tissues, may still show the
effects of freezing artifact.  These effects may be seen more in cartilage
with its higher water content, but give the LM samples a try. You may
salvage part of the study.  Then send the student out for samples and insist
he/she reads up on and understand the damage freezing/thawing can have on
fixed samples.  

 

I suggest, in lieu of sending your student to the Arctic, that he/she reads
the excellent discussion of freezing artifact by Charles Scouten (Myneurolab
website) , titled Tips and Techniques: Freezing Biological Samples to
prevent this from happening again.   A supporting publication, cited by
Scouten, is Jongebloed, W.L., Stokroos, D.,  Kalicharan, D., and Van der
Want, J.J.L.  Is Cryopreservation Superior Over Tannic Acid/Arginine/Osmium
Tetroxide non-Coating Preparation in Field Emission Scanning Electron
Microscopy.  Scanning Microscopy 13: 93-109, 1999.  This paper might help
clarify what damage occurs for SEM even though you might be doing TEM (?).


 

I will be happy to send the article in a separate email if you want it.  

 

It would be interesting to have follow up on your results, so keep us
posted. 

 

Good luck

 

Gayle Callis 

HTL/HT/MT(ASCP) 

 

    
 
 
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