NetNotes
Edited by Thomas E. Phillips University of Missouri
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from January 1, 2011 to March 2, 2011. 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: hexamethyldisilazane (HMDS) I’m looking for a reference to explain just how/why HMDS works
to dry specimens for SEM observation. T ere are plenty of references showing that it does, in fact, work and produces good results in many cases, but I have yet to fi nd an explanation as to the “why.” Is it that HMDS has the right physical properties (surface tension) to evaporate without causing drying artifacts? Alternatively, is there something about the silanization of the sample that helps make it more robust, or both, or am I completely lost (quite likely)? Bryan Bandli bbandli@d.
umn.edu Wed Feb 2 I believe it has to do with very low surface tension. It also does
not work well across the board. I understand that it works well with insects, but one of my sons did a science fair project comparing dried, freeze-dried, and HMDS treated green pepper. In his case the HMDS didn’t appear to be any better than drying. His best results were from plunging small pieces into acetone cooled with a Peltier cooler, and then transferring to another Peltier cooler in a vacuum evaporator until dehydrated. He was not able to compare it to critical point dry prep as we didn’t have the requisite CO2. Obviously, the plunge freezing was not done at typical temperatures, so there was probably ice damage in those samples, but they looked the best of the bunch. Ken Converse
kenconverse@qualityimages.biz Wed Feb 2 Drawing from my cleanroom experience. . . . HMDS is used as a
photoresist adhesion agent for Si wafers. It binds to hydroxyl groups. It makes sense to think of as having mordant properties, strengthening the specimen with Si. T e low surface tension also plays an important role.
http://www.transene.com/hexa.html. Edward Basgall ejb1176@
gmail.com T u Feb 3
Specimen Preparation: SEM sample storage One of our EM lab users asked me a question: what is the best way
to store SEM samples? I know you’ll all say that the best is to process and coat fi rst. He wants to store them in 2% glutaraldehyde and process later since he does not have time now. Dorota Wadowska wadowska@
upei.ca Wed Feb 2 To my experience, specimens can stay in glutaraldehyde for
an indefi nite time. I have processed samples aſt er >3 years stored in glutaraldehyde inside the fridge, and they looked the same as others being processed within a few days. In rare cases, I’ve seen mushrooms developing in samples that stayed only a few weeks in glutaraldehyde. T ere, I suspect that the glutaraldehyde was old, samples were dirty from the beginning, or some other unidentifi ed factor was present. If you process and coat the specimens, I think it is better to view them soon, otherwise they can aff ected by humidity and the morphology deteriorates. In conclusion, I think it is not a bad idea to have your samples staying in glutaraldehyde until you have the time to view them. Yorgos Nikas
eikonika@otenet.gr Wed Feb 2
62 Glutaraldehyde can deteriorate over time and produce a white
precipitate that could coat the specimens. For long term storage it is not a good idea. Ralph Common
common@msu.edu Wed Feb 2 Store for how long? What kind of specimens? I have stored
soſt tissue samples for a couple of days in glutaraldehyde with no problems, and crustaceans with well sclerotized cuticles for several days, but lightly sclerotized crusties and body parts don’t like it. Note! Watch the pH! T e fi x can cause dissolution of Ca2+ in the crustacean exoskeleton. When I had to process, store, then transport samples from Antarctica to Chicago, I ran them up into 100% Pel-Dri (back when it was allowed) or 100% HMDS. T is worked. T e samples fi sh lateral-lines and lateral-line end organs, and they compared well to freshly fi xed, processed, and dried end-organs collected in Lake Michigan. T e sample vials where completely full of fl uid, with a 10:1 or 20:1 fl uid:specimen volume ratio. But the best way is to fi x, process, dry, mount, and stick in a desiccator. Don’t coat. T e expansion and shrinkage of the samples when moving them from the desiccator to the SEM and back can cause fi ne cracks in a sputter-coated metal layer. Coat when ready to examine. Philip Oshel
oshel1pe@cmich.edu T u Feb 3
Immunocytochemistry: post-embedding FluoroNanogold labeling for TEM Some of our antigens are diffi cult to label in mammalian fat cells
and muscles, such as particular lipid droplet proteins. I have tried several methods of antigen unmasking and probe maintenance in post-sectioning fl uoronanogold labeling, including absence of uranyl acetate in the LR-White embedding medium, reduction of osmium post-fi xation concentration to reduce etching propensity of
silver-
enhanced gold probes, antigen unmasking with oxidizing (sodium metaperiodate) and reducing (sodium borohydride) agents and Tris base pH 10. I have reduced my glutaraldehyde concentration to 0.10% and maintained paraformaldehyde at 2%. Pre-embedding fl uoronanogold labeling appears to work with the absence of glutaraldehyde in the fi xative, however the ultrastructure is quite compromised, as one would expect by TEM. I have been thinking now of using LR-Gold, until I hear from some experts on any other suggestions. Vickie Kimler vakimler@
med.wayne.edu Mon Jan 24 I had a similar situation and had successfully used pre-embed
labeling with FluoroNanogold. In the post-section fl uoronanogold labeling, there might not be enough cross linking of your proteins to keep them stabilized especially during the dehydration and embedding steps. Antigen unmasking might not be the issue there but rather the proteins have been extracted. From what I recall lipids rely on osmium tetroxide and uranyl acetate fi xation. Assuming you have abundant amount of these proteins, if you have already tried the gradual low temperature approach during dehydration and resin infi ltration steps and there is still no labeling, I would try another approach. You mentioned that pre-embed fl uoronanogold labeling worked but the ultrastructure quality was quite compromised. T at
doi:10.1017/S1551929511000319
www.microscopy-today.com • 2011 May
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