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modifications we introduce. Te formaldehyde/glutaraldehyde mix is used for decades now and frankly I won’t dare stand up against the huge amount of literature produced with the classical methods and claim that they introduce systematically significant artifacts. What you can say is that you lose some structures, that you don’t see everything. Tis is right (and this is confirmed by the use of cryofixation).


Te facts are that formaldehyde is a fast-penetrating fixative, but its fixation is weak and (at least partly) reversible. Tis is why it is oſten used in association with glutaraldehyde, which is slower but stronger. Both together generally offer a fixation of quality. However I have a problem with the original message of Pete and specifically with the part “artifacts . . . in general.” Tere is no protocol that works “in general.” All protocols must be adapted to the specimen. In general, with protocols well adapted for the specimen, formaldehyde fixation does not produce membrane blebbing artifacts. However it may well be that a general protocol is not suited for Pete’s specimen. Also, generally formaldehyde fixation is completed by an osmium post-fixation step but this was not stated. Not knowing with which specimen Pete is working (and how) it is hard to guess what could be wrong, but I wouldn’t be surprised if it had to do with osmoticity. Stephane Nizets nizets2@yahoo.com Mon May 2 An early paper showing membrane induced artifacts resulting


from aldehyde fixation: Hasty & Hay (1978) Freeze-fracture studies of the developing cell surface. II. Particle-Free Membrane Blisters on Glutaraldehyde-Fixed Corneal Fibroblasts Are Artefacts. J. Cell Biology 78:756–768. Tere are other papers showing the dramatic change in


morphology to mitochondria following aldehyde fixation compared to those prepared by high pressure freezing or quick-freezing using the metal mirror approach. I don’t have a copy to double check but I believe that is shown in Terracio L, Bankston PW and McAteer JA (1981) Ultrastructural observations on tissues processed by a quick-freezing, rapid-drying method: Comparison with conventional specimen preparation. Cryobiology 18(1):55–71. Here is a study showing aldehydes cause dramatic changes in endosomes: Murk JL, Posthuma G, Koster AJ, Geuze HJ, Verkleij AJ, Kleijmeer MJ, Humbel BM. (2003) Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography. J Microsc. 212(Pt 1):81–90. Tom Phillips phillipst@ missouri.edu Mon May 2


Microtomy:


long-term storage of blocks Does anyone have any opinions on whether resin blocks


deteriorate over time (i.e. decades), and whether some storage options (airtight, low temperature . . . ?) are more appropriate than others? Tis has implications for type specimens deposited in museums, where the primary description of a species is based on TEM. Single-celled eukaryotes oſten have no other useful type material. Anecdotally, preservation of ultrastructure in some blocks from the 1960s has deteriorated since they were looked at in the 1980s—but it’s unclear if this is a general pattern. Giselle Walker giselle.walker@anatomy. otago.ac.nz Mon Apr 4 Are you thinking of deterioration of resin over time?


Polymerization may not be completely over and done with within a day or two, but continue more and more slowly during storage. Also UV light exposure should probably be avoided. All this would make the blocks more brittle over time, by reducing chin length or breaking bonds. But there is also the risk that any of the used chemicals (e.g. OsO4) is not completely inactive or thoroughly washed out before embedding. And there is not much one can do aſterwards. I would


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therefore choose conditions that reduce reaction speed (Q10) in general, i.e. relatively low temperature storage away from direct light. Jan Leunissen leunissen@aurion.nl Mon Apr 4 I just sectioned blocks from 1976 two weeks ago. Te blocks


cut fine, with the exception that some areas had not been infiltrated properly when they were initially processed. Te tissue was of sea slug that was rare and irreplaceable. We got the information from the blocks that we needed. Te plastic was Epon-araldite. Tissue preservation was perfect in properly embedded areas, and the plastic was stable in the beam. If the samples are prepared properly in the first place, they are permanently archived in the plastic for a long, long time. Store the blocks in a climate-controlled area. Te blocks I cut were stored in someone’s attic. I was surprised that they survived the harsh heat and cold in that environment! I have sections that I cut, still on grids, from 1974, that I can put in the TEM and photograph today. If the sections are stored properly, they are archival, too. Tey are not on carbon or Formvar films, just bare copper grids that have been kept in a low humidity environment all this time. I have re-scoped them several times before. It’s been my experience that tissue deterioration in blocks comes from improper dehydration or infiltration during processing. If the samples were run up properly in the first place, the tissue is archival. It is possible, and I have seen this, to have a processing schedule that is barely usable, that just gets a person by. Tis produces tissue where the osmium is not fully washed out, or the tissue is not fully dehydrated, and the blocks are either soſt or brittle. You may be able to cut the tissue initially, but the blocks deteriorate with time. Well processed tissue will not do this. Te processing problems usually occur when trying to process tissue that is too large, or when running something up too fast, or when using old chemicals. Tat, at least, has been my experience. You may have come to similar conclusions. Epoxy blocks commonly used for E.M. should be archival. Lowicryls and LR White and Gold may pick up moisture with time and deteriorate. Tey should also be protected from UV light, since most of the work with these resins is for immunogold labeling, and antigenic preservation is the goal. I always remove my blocks from block molds. I don’t know if the plastic from the mold will interact with the tissue or epoxy in the block with time. Ed Haller ehaller@health.usf.edu Tue Apr 5 Several years ago I sectioned blocks that were about 20 years old


and they were perfect. Tese were ones that had not been previously used or trimmed. Tey had been kept in thin cardboard pill boxes inside 8 × 10 inch Kodak photographic paper boxes in a glass door cabinet. Humidity in the summer in Philadelphia is usually very high so I’d say that the lab ran about 80% even with the air conditioning on. Temperature in the lab would range from near 65 most of the winter (trouble with heating system in our ancient building) to high-70’s in the summer. To my knowledge these blocks would have been made aſter I had run out of Epon 812 (original formula from Shell, for the younger members of our Listserv) but they just may have been embedded with the “good stuff”toward the end of my supply. Te blocks were cured at 60°C for a minimum of 48 hours if not over a weekend. You mentioned that your blocks were from the 60’s. Tat was still a time when Epon 812 was still the embedding of choice for many labs but there were also many other embeddings being used like Araldite/Epon mixtures with which I am unfamiliar. Patricia Stranen Connelly connellyps@nhlbi.nih.gov Tue Apr 5


Microtomy: slide labels I was wondering what others may be using to make their labels for


glass slides permanent and insoluble to ultrasonic cleaning in ethanol. I’ve seen some slides where someone had labeled with a Sharpie, then


www.microscopy-today.com • 2011 July


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