Agar/sucrose cryo-embed

From:Karen Larison <>

<!doctype html public "-//W3C//DTD W3 HTML//EN"> <html><head><style type="text/css"><!-- blockquote, dl, ul, ol, li { margin-top: 0 ; margin-bottom: 0 } --></style><title>Agar/sucrose cryo-embed</title></head><body> <div>Connie,</div> <div><br></div> <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> <div><br></div> <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> <div> </div> <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> <div><br></div> <div>Karen in Oregon</div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <div><br></div> <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> <div><br></div> </body> </html>
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