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Protistans are so variable, you really have to experiment, or get a known good protocol from someone who does your particular organism - Trichomonas tenax , here. From processing euglenoids, we fi nd even congeneric taxa can vary greatly in their processing requirements, for both SEM & TEM. Phil Oshel oshel1pe@cmich.edu Fri Jul 17 Protists are notoriously variable things; diff erent but closely related strains can respond very diff erently to the same fi xation protocol. So you might get good results the fi rst time you try, or you might have to experiment. To answer your questions in order: I usually use a CPD for SEM prep, and I’ve gotten good results with that. However, there are a few diff erent types of CPD, some of which require careful operation. T us, the results may depend both on the type of CPD and the operator. I’ve also used tert-butanol in a freeze-dryer with adequate results, although I’ve gotten better results in some cases with the CPD. I’ve never used HMDS but I’ve heard very good things about it. For mounting, I’ve used both poly-L-lysine-coated coverslips (the round 12-mm types, which unfortunately tend to be expensive) and Isopore fi lters. Give the cells about an hour on the coverslip and they’re stuck. Isopore fi lters don’t require any treatment at all: I fi x my cells in a Petri dish and just plunge them through the fi lter and that’s enough. Of course you want to be careful not to apply back-pressure when removing the cartridge, handle anything with cells adhering to it gently, and keep it wet! A few seconds at most out of ethanol is fi ne, though. As for TEM, I ‘trap’ my cells in 2% agarose aſt er fi xation. I do this by pipetting a shallow puddle of molten agarose onto a fresh slide (use a transfer pipette or a cut-off Gilson: narrow pipettes will clog!) and then pipetting 1–10 µl of concen- trated fi xed culture directly into the puddle, trying to suspend the cells directly in the middle of the water (agarose?) column. Aſt er the agarose sets, I cut out a ~1-mm cube around the cells, and do my dehydration and embedding with the cube. I’ll typically get 4–6 cubes from a single fi xation. One nice aspect of this protocol is that, for the resin-containing stages, the sample will fl oat if it’s not ready for the next change! I also have colleagues who instead spin down their cells and dehydrate and embed them ‘free’ in an Eppendorf tube, but my cultures are usually too sparse to deal with the amount of material one loses in that procedure. Aaron A. Heiss aheiss@amnh.org Sat Jul 18


Specimen Preparation: paraffi n sections for TEM


I have one user, who wants to carry out TEM analysis of his Histology samples. He wants only histology slide stained with H&E. Has anyone done this kind of work before? Kindly give the best suggestion from your experience. Ravi T akkar ravi.thakkar369@gmail.com Fri Aug 28


If you mean preparing an H&E section for TEM, I suspect you are in for a disappointing result. I haven’t done it for an H&E section but have done it for an unstained paraffi n section. I osmicated the section, infi ltrated with resin and polymerized resin on the surface of the slide. I then popped it off the slide using liquid nitrogen and mounted en face on an epoxy block so I could section it. T e tissue was recognizable in the TEM but the quality of the tissue preservation was terrible. It looked vacuolated. I am always amazed how “acceptable” tissue fi xation looks like at the LM level but how bad it looks like once you go to TEM. TEM is tedious and demanding enough with optimally fi xed tissues so it is best to handicapping your chances before you even start. Unless the tissue is something incredibly rare, it is worth repeating with proper fi xation and embedding for TEM. Tom Phillips phillipst@ missouri.edu Sat Aug 29 It is not oſt en that someone, via the MSA Listserver, requests such measures/procedures. T is is probably due to the decrease of med.-diagnostic EM-ists (unfortunately many medico-diagnostic


52


EM-Labs have been cut and more will become extinct) out in the wild. He or she must be a young pathologist or clinician, I guess. Seconding 100% Prof. Phillips’s opinion (and making no diff erence between an H&E stained=deparaffi nized or unstained paraffi n section): “Garbage in, Garbage out”. If the tissue is initially fi xed poorly then poor / bad preservation of ultrastructural detail will be the “natural” consequence. But sometimes careful and sophisticated reprocessing of - selected areas of - either tissue from whole paraffi n blocks or also a H&E-section only can yield some interesting and unforeseen result in terms of diagnosis (viral, bacteria, etc. etc., see also below). So in the end: It always will depend on the circumstance and task of the study whether such a re-embedding/reprocessing (which might be sumptuous and/or a bit complex to accomplish) will yield something of value (e. g. especially something diagnostic) - worth to be documented. In my 35 years EM-career I have done about 100–150 re-embeddings (from paraffi n blocks as well as from deparaffi nized H&E and/or pre-embedding IHC-sections) and the scientifi cally useable yield was estimated at about 65–70% (esp. for evaluating the preembedding immunolocaliztion of markers in DAB-treated immunohistochemistry sections). Some examples for your convenience: Estrada, J.C. et al., “TEM of Paraffi n-Embedded H&E Stained Sections for viral Diagnosis (an Unusual Papovavirus Case),” Microscopy Today , Sept. 2005, pp. 22–24. See also, for example, J. Burns, “Preparation of thin epoxy resin sections from thick sections of paraffi n-embedded material,” J Clin Path 23(7) (1970) pp. 643–45; or Van den Bergh Weerman M.A., Dingemans, K.P. Rapid, “Deparaffi nization for electron microscopy,” Ultrastructural Pathology 7 (1984) pp. 55–57; or S. Widéhn, and L.-G. Kindblom, “A Rapid and Simple Method for Electron Microscopy of Paraffi n- Embedded Tissue Ultrastructural Pathology,” 12 (1988) pp. 131–36; or Lighezan, R. et al., “T e value of the reprocessing method of paraffi n-embedded biopsies for transmission electron microscopy,” Romanian Journal of Morphology and Embryology 50(4) (2009) pp. 613–17. Wolfgang Muss w.muss@salk.at Mon Aug 31 I know you just asked if we’ve already done this kind of work, not what we think about it. However I must second both T omas and Wolfgang. Technically it is not such a big challenge but we like to know if what we do makes sense somehow. You rarely give yourself the hassle of TEM preparation because it is cool. Usually one has a precise purpose in mind. If your colleague/client wants fi ne morphological information, he will be very disappointed. Stephane Nizets nizets2@ yahoo.com Tue Sep 1


Specimen Preparation: preparing frozen insects for TEM


We’re interested in processing moths for TEM, but the moths have been stored in a 0°F (-18°C) freezer for a few weeks or months. Will it still be possible to immerse these moths in glutaraldehyde and process them for conventional TEM? I understand the ultrastructure may not be optimal, but has anyone had success processing frozen insects for TEM? Joe Mowery joseph.mowery@ars.usda.gov T u Aug 20 Summary (you don’t need the details to take a decision): “Not optimal” is mildly put. Leaving biological material unfi xed at −18°C (it is probably the worst method to preserver biological material) and expecting to see anything meaningful in TEM is not realistic. If you want to use TEM, you are interested in fi ne morphology right? Well you won’t get fi ne morphological information this way. Details (if you want to know why): 1) Upon freezing and especially slowly freezing like it happens when you put a specimen at −18°C, the water in the tissue and cells crystallizes. Nice spikey crystals. T e crystals don’t care about the biological structures (like membranes); they grow


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