FW: TEM/LM: Problems with cat retinal tissue

From:"Monson, Frederick C."

Morning Sergeant,
	This message was sent to the MSA listserver a while back and I saved
it for obvious reasons.

Hope it helps,


Frederick C. Monson, PhD
Center for Advanced Scientific Imaging
Mail Drop:  Geology
West Chester University
West Chester, PA, 19383
Phone/FAX:  610-738-0437 

-----Original Message-----
From: Sherwood, Margaret [mailto:MSHERWOOD@PARTNERS.ORG]
Sent: Friday, August 23, 2002 12:19 PM
To: 'Tindall, Randy D.'
Cc: 'microscopy@sparc5.microscopy.com'
Subject: RE: TEM/LM: Problems with cat retinal tissue 

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I have been working with rabbit retina that has been lased at different
pulses/pulse durations, looking for damage in the epithelial layer.  I have
had excellent preservation of the retina (the normal adjacent areas as well
as the damaged areas).  I recently purchased a microwave to do fixation and
processing, and ran a couple of duplicate samples to compare.  I also got
good results.

Because I haven't found a really inclusive protocol for microwave processing
(I couldn't get my blocks to polymerize via the microwave), I resorted to
using a conventional oven for that stage.


1.  The eyes were injected with Karnovsky's Fixative( usually, sometimes
with 4% glut) after lasing and left overnight in the cold (this is to
maintain the integrity of the structures).

2.  The posterior segment of the eye is cut away and then put in fresh 4%
Glutaraldehyde in 0.1M Cacodylate Buffer (pH 7.4) and stored in the cold.
Note:  these segments can remain in the cold indefinitely (i.e. two weeks).

3.  The vitreous fluid is teased away gently so as not to disrupt the
underlying retinal structures.  The lased areas are dissected away from rest
of posterior segment using an  ophthalmological knife*.  Again, to avoid
disrupting the structures.

4.  The lased segments (@1mm square) are put into 0.1M Cacodylate Buffer (pH
7.4) until processing.


5.   3 X 15 minutes  -  0.1M Cacodylate Buffer
6.   Overnight fixation in 2% OsO4 in cacodylate buffer (in the cold)
7.   2 X 15 minutes  -  0.1M Cacodylate Buffer
8.   Dehydration:  25% ETOH, 50% ETOH (2 X 15 minute changes each); 70% ETOH
(2 X 30 minute changes) This is a good stopping point:  then I leave them in
a 3rd change of 70% overnight in the cold.  
9.   2 X 15 minutes - 95%  ETOH
10. 4 X 15 minutes - 100% ETOH
11. 2 X 15 minutes - propylene oxide
12. 2:1 propylene oxide:epon (overnight or minimum 2 hours)
13. 1:2 propylene oxide:epon (overnight or minimum 2-3 hours)
14. 100% epon - all day and then embed at end of day.
15. Polymerize in 60 degree oven overnight (18 hours).  (I have actually
left them for 2 days in the oven and the blocks have been fine.  I hesitate
to leave them too long; the blocks then get brittle)

I order my embedding components from Tousimis (Rockville, MD):
EPON 812 - Cat# 3131
DDSA	     - Cat# 3123
NMA	     - Cat# 3143
DMP-30     - Cat# 3103A

I store components under hood at rm. temperature and freeze aliquots of
complete epon.  I use aliquots for the interim steps (i.e.with propylene
oxide) but make up fresh epon for embedding.   I make up the epon on a stir
plate, stirring after addition of each component. I then put it under vacuum
to get rid of excess air bubbles.

I realize everyone's protocol is slightly different (judging from the
responses you've received), but as long as you maintain the structure of the
retina, you should have no problem with vacuoles or tears in the tissue.

Hope this helps.
Now...if you could send me a protocol for microwave processing, I would
greatly appreciate it!  

Peggy Sherwood
Lab Associate, Photopathology
Wellman Laboratories of Photomedicine (W224)
Massachusetts General Hospital
55 Fruit Street
Boston, MA 02114
617-724-4839 (voice mail)
617-726-6983 (lab)
617-726-3192 (fax)

> -----Original Message-----
> From:	Tindall, Randy D. [SMTP:TindallR@missouri.edu]
> Sent:	Wednesday, August 21, 2002 10:14 AM
> To:	microscopy@sparc5.microscopy.com
> Subject:	TEM/LM: Problems with cat retinal tissue 
> ------------------------------------------------------------------------
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> -----------------------------------------------------------------------.
> Dear Listers,
> We are having some difficulty with getting good thick sections from feline
> retinal tissue. The sections look somewhat uneven in thickness (wavy), and
> in the nerve fiber layer and at the level of the inner limiting membrane
> there is vacuolization.  There are also tears in some areas.  Our standard
> processing procedures use 2% gluteraldehyde/2% paraformaldehyde in 0.1M
> cacodylate buffer, 1% osmium post-fix, 1% uranyl acetate en bloc stain,
> ethanol dehydration, and Spurr's/EPON resin.  We are currently trying 2.5%
> gluteraldehyde in cacodylate (at 0.1 and 0.17M), as well as combining
> microwave fixation with our standard procedures.
> Our current procedures---microwave and otherwise---work very well with
> mouse retinal tissue, but cats are being contrary.  If anyone has any
> tried and true techniques, I'd be very grateful to hear about them.  It
> could save us tons of time and frustration as we develop a new set of
> protocols.
> Thanks very much.
> Randy  
> Randy Tindall
> EM Specialist
> Electron Microscopy Core Facility
> W122 Veterinary Medicine
> University of Missouri
> Columbia, MO  65211
> Tel:  (573) 882-8304
> Fax: (573) 884-5414
> Email: tindallr@missouri.edu
> Web: http://www.biotech.missouri.edu/emc/

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