NetNotes
Edited by Thomas E. Phillips University of Missouri
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from May 1, 2010 to June 30, 2010. Complete listings and subscription information can be obtained at
http://www.microscopy.com. Postings may have been edited to conserve space or for clarity.
Specimen Preparation: liver
T ank you for those many that have tried to help with my problems
with fi xation and embedding of liver samples. T ere have been many good suggestions but so far none solves my problem. I would like to be a bit more specifi c in describing our protocol just in case someone might think of another possible cause. To recap, we have had consistent problems with extremely brittle blocks when cutting thick or ultrathin sections of liver. T e tissue (not the surrounding blank Epon) fractures like glass, even during trimming with a double edge razor blade. We use the same protocol we use for the many other tissues that we embed well, so this problem is specifi c to liver. Our protocol includes primary phosphate buff ered fi xative (1.5% glutaraldehyde + 1.5% paraformal- dehyde + 0.05% tannic acid) followed by 1% OsO4 (with appropriate buff er rinses), dehydration in ethanol to 100%, a rinse in propylene oxide and infi ltration and embedding in Spurr’s resin, mixed according to Ellis. We have also embedded in Epon 812 substitute with similar brittleness. As I say, we use this protocol successfully for all other tissues. One suggestion received is that there is some water leſt in the sample which makes embedded tissue in Spurr’s very brittle. I am sure our ethanol, propylene oxide and media is dry, but is it necessary to dehydrate liver longer than other tissues? T e tissues are initially fi xed in another laboratory and then sent to us. T e tissue pieces are about 2 mm × 2 mm but were fi xed in two changes of primary fi xative for several hours each; then stored for a couple of weeks, then sent to us. Once in my laboratory, they are cut down so that one face is no larger than 0.5 mm. T en they are osmicated, dehydrated and embedded. Could either the initial excessive size of the tissues, excessive fi xation time or storage in buff er be a factor? Does anyone have another suggestion? If you work successfully with liver, would you mind sending a protocol? Doug Keene
drk@shcc.org Mon May 3 Well, I hesitated to respond, but when we see this problem
in plant tissues, it’s the embedding—resin infi ltration, that’s the problem, not fi xation or dehydration. You may want to try infi ltrating more slowly—with smaller resin increments, or for somewhat longer than whatever protocol you’re using at present. In plants, the problem is almost impermeable cell wall (i.e. extracellular matrix) layers in the tissue that either prevent or greatly slow down infi ltration of resin polymers. Are there any structures in liver that might slow polymer diff usion? In algae, for example, some ECM components are very impermeable, especially aſt er osmication, and to avoid collapsing algal zygotes in some species (because solvent can diff use out rapidly but resin can diff use in only slowly), a colleague used to increment the resin by 2% per day over the beginning and end 10–16% steps of infi ltration, with larger steps in the intermediate concentrations—it took a month to infi ltrate. It did mean she had to do this only once and had perfect TEMs.... T at’s an extreme example, of course! Rosemary White
rosemary.white@csiro.au Mon May 3 I also hesitated, knowing that it is unlikely that I might have any experience beyond that of yours or John’s. Since you explained
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more, though, this has moved into a mystery category, so ... (1) I do have some successful experience processing liver. It was ~15 years ago, but I remember the protocol fi rmly and also that there were no trouble with blocks. I studied mitochondria as an undergraduate, and so the ultrastructure was good too. 5% glutaraldehyde in 0.1 M sodium phosphate buff er for 2–3 hr. Bigger pieces were put in this fi x and cut down to EM size by slicing with 2 sharp razor blades. Aſt er some short time, the sample was put in the refrigerator, and all steps from that point on done in the refrigerator, until acetone. 1% OsO4 in same buff er. Buff er washes. 50% ethanol, 60%, then overnight in 70% with 1.5% uranyl acetate. Next day, 80%, 90%, 2x96% (that’s what it said on the bottle, pure ethanol but not dried). T en 2x acetone, 15–30 min each at room temperature. T en 3:1, 1:1, 1:3 acetone:Epon (original Fluka Epon). T en the barbaric step of 12-24 h at 37°C in pure Epon, then 48 h at 60°C. T e liver came out excellent. You can see that this protocol is not optimal in terms of infi ltration, and it did get me in trouble with some other objects (like holes in bacteria), but not with liver. A higher percent of glutaraldehyde (4–5%) has always served me very well. For the kind of work I was doing then, I never saw any advantage adding formaldehyde to glutaraldehyde. Fine detail of the mitochondria always came out worse. (2) Doug, is that human liver? What I did was mouse and rat, of course. With humans, who knows what they’d been eating and drinking. (3) One thing that immediately caught my attention in your second post— that someone else was doing the fi xation. Could it be something they do there? (4) Finally, if you can’t solve it but still must trim and cut it, may I recommend my favorite blades? It is # 71930 from EMS catalog, Solingen Long Blades. Much harder and sharper than anything else. T ese will make a cleaner cut. Vlad Speransky vladislav_speransky@
nih.gov Tue May 4 I had a similar problem last week with some nerve tissue. It was
embedded in EMbed-812 (Epon alternative). However, the tissue was brittle and crumbled only in some areas and I am quite certain that it is related to poor fi xation in those areas. I suggest that you participate in the fi xation at least once and make sure that the tissues blocks are minced quickly and with minimal mechanical distortion to a size about 1 mm × 1 mm. T e prolonged storage is not helpful but may be unavoidable. Let us know if you fi nd the solution. Larry Ackerman
larry.ackerman@
ucsf.edu Mon May 10
Specimen Preparation: silicon microspheres I mostly work with biological samples. I have a researcher who
is working with crystals formed from ~12 nm Si microspheres; this is somewhat like opal, I think. It forms nice crystals that are easily cleaved with a little pressure from forceps, etc. Lacking a fi eld emission SEM, we have shadowed the surface of the crystals and fi nd a nice hexagonal packing. T ey are a bit too small to be able to cleave in a controlled way in a chosen plane. T e researcher would like to know if the crystals have
doi:10.1017/S1551929510000775
www.microscopy-today.com • 2010 September
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