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<div>Connie,</div>
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<div>You don't need to let the agar infiltrate. In fact you
have to work rather quickly to orient the embryo before the hot agar
re-solidifies. Here's the formula:</div>
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<div><font face="Helvetica" size="+2" color="#000000">1.5 gm agar
(use bacteriological grade)</font></div>
<div><font face="Helvetica" size="+2" color="#000000">5.0 gm
sucrose</font></div>
<div><font face="Helvetica" size="+2" color="#000000">100 ml
distilled water</font></div>
<div><font face="Helvetica" size="+2"
color="#000000"><br></font></div>
<div><font face="Helvetica" size="+2" color="#000000">Place
ingredients in erlenmeyer flask and set flask in a beaker of
water. Stir on magnetic stirrer and then heat until
dissolved. Solution should be clear. Store in 20 ml
scintillation vials at 4C. Melt in microwave oven or in hot
water bath. Cool to 40C for use.</font></div>
<div><font face="Helvetica" size="+2"
color="#000000"><br></font></div>
<div>We transfer the washed embryo to a small peel away mold, remove
all the liquid, poor on the hot agar (about 40C), and work very
quickly to orient it. The bottom of the mold becomes the top of
the block. You wait for 10-15 minutes until the agar-sucrose is
solidified, and then we gently pry it out of the mold, and sink in
30% sucrose overnight in the frig. It's easy to trim the block
with a razor blade, both before you freeze it and after you begin
sectioning. This particular agar formulation requires a cryostat that
holds its temperature well (+/- 1.5 degrees). We cut it at
around -18C - 20C.</div>
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<div>If your cryostat doesn't hold the temperature well, you can use
a combination of agar and low-melt agarose. The low-melt
agarose causes the block to be more tolerant of temperature
variations, but produces blocks that don't ribbon quite so
nicely. Use 0.9 g agar and 0.9 g low-melt agarose (instead of
the 1.5 g agar) in the protocol if cryostat temperature variation
proves to be problematic.</div>
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<div>Karen in Oregon</div>
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<blockquote type="cite" cite>At 11:17 AM 5/17/01 -0800, Karen Larison
wrote:<br>
>We first embed them in a<br>
>melted agar-sucrose matrix, which allows us to orient them
very<br>
>precisely within the block for serial sectioning. <br>
<br>
Karen,<br>
What is the formula for the agar and sucrose? I am interested
in trying<br>
this. Do you need to let the tissues infiltrate in the agar for
a while?<br>
<br>
Connie McManus<br>
<br>
<br>
We then let the<br>
>block solidify and then sink it in in sucrose before
sectioning. The<br>
>nice thing about these agar blocks is that they ribbon
nicely. You<br>
>can cut a series of sections with the antiroll plate, then use
a<br>
>brush to straighten them out, and then pick them up. Using
this<br>
>method, we are better able to position these tiny sections on
a<br>
>slide, we are less likely to experience "leaping"
sections, and we<br>
>can get 70-100 sections on one slide in very short order!<br>
><br>
>We also use Tissue Freezing Medium (made by TBS, available
from<br>
>Fisher Scientific) to adhere our agar block to the chuck, and
find<br>
>that this curls less, and appears to have less static problems
than<br>
>O.C.T.<br>
><br>
>Karen in Oregon<br>
><br>
><br>
><br>
><br>
><br>
>> Has anyone encountered a phenomenon that I
occasionally get<br>
>>with cryostat frozen sectioning. I assume it is due to
static<br>
>>electrical charges? the sections tend to "leap"
from the knife edge<br>
>>onto the slide, the slide may be some several centimeters
from the<br>
>>section/knife holder.<br>
>>There is no logic or consistency to this, irrespective of
tissue,<br>
>>fixation, temperature, type of slide; but if it is going to
happen then<br>
>>you can be sure it is when cutting a tiny piece of tissue
where<br>
>>every section is required! It will often disappear as
suddenly as it<br>
>>started, sometimes quite impressive when a section fly's
several<br>
>>cms. to the slide and occasionally lands flat and crease
free!<br>
>>Any idea's?Oh, and I don't wear nylons (not in work
anyway).<br>
>>Terry.<br>
>>Terry Hacker,<br>
>>Medical Research Council,<br>
>>Harwell,<br>
>>Didcot,<br>
>>Oxfordshire, OX11 ORD<br>
>>01235 834393 x360<br>
><br>
><br>
><br>
Connie McManus<br>
Veterinary Diagnostics Lab<br>
Utah State University<br>
Logan, UT<br>
USA</blockquote>
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