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NetNotes


Edited by Thomas E. Phillips University of Missouri


phillipst@missouri.edu


Selected postings from the Microscopy Listserver from May 1, 2017 to June 30, 2017. 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: gold labeling protein complex


I have a client who will be submitting outer surface proteins (OSPs) to resolve by TEM. He plans to use gold conjugated to the primary antibody, which will express an antigen on these OSPs. Does anyone have a protocol or know of any older articles related to this? Some concerns and questions I have are attachment of the OSPs onto a Formvar (or Parlodian) grid; using a routine immuno-electron microscopy procedure or a revised protocol; using a routine negative stain (uranyl acetate or phosphotungstic acid) or would low angle shadowing work more effi ciently? Mary Ard maryard@uga.edu Wed Jun 21 For uniform spreading of protein particles on the grid, it is best to have a glow-discharged carbon fi lm on top of your Formvar or Parlodion. If that is not an option, bacitracin solution oſt en helps, or even BSA. T en you can use any basic immunoEM protocol, which you can obtain from any major supplier or perhaps from community members here. Use minimum blocking fi rst, to establish some binding, then you can include extra blocking steps or higher concentrations, if there is an objectionable background. T is usually works quite well for a protein laying on a grid, with no obstacles, like when it is somewhere deep in the cell. What species is the primary antibody from? If possible, I always preferred to use Protein A-gold as a secondary probe. Much cleaner and more precise. It is best with rabbit polyclonals but will not work with most mouse monoclonals. Finally, the contrasting step - go with PTA (or ammonium molybdate) over UA: not as dark and spreads easier. Let me know if you need a sample protocol. Vlad Speransky vlad_speransky@tedpella.com Wed Jun 21


Specimen Preparation:


ethanolic phosphotungstic acid staining I am trying to fi gure out what part of the cellular organelle or molecules the ethanolic phosphotungstic acid (E-PTA) binds to show the electron dense staining? I have found that it’s commonly used for visual- izing phospholipids or nerve ending. However, I do not know if E-PTA binds to lipids molecules or membranes. Does anyone know exactly what E-PTA binds to? Kyoung Jo kyoung.jo@rockets.utoledo.edu Mon Jun 5 I guess you are referring to the ethanolic phosphotungstic acid (E-PTA) staining technique, which was introduced by Gray (1959). [cf: Stains and Cytochemical Methods , M.A. Hayat (ed.) 1993] “Ethanolic Phosphotungstic Acid,” ch. 8, p. 284, where you can fi nd a collection of stained and unstained material/tissue components, predominantly of/regarding nerve synapses. Perhaps you can fi nd some or more of the information you are looking for in:


Hu et al., “Assembly of Proteins to Postsynaptic Densities aſt er Transient Cerebral Ischemia,” J Neurosci 18 (1998) 625–633. FE Bloom and GK Aghajanian, “Cytochemistry of synapses: a selective staining method for electron microscopy,” Science 154 (1966) 1575–1577.


64


FE Bloom and GK Aghajanian, “Fine structural and cytochemical analysis of staining of synaptic junctions with phosphotungstic acid,” J Ultrastruct Res 22 (1968) 361–375. Also, you can fi nd hints on the nature of material that is stained (or less or not stained) by E-PTA in: Horowitz and Woodcock, “Alternative staining methods for Lowicryl sections,” J Histochem Cytochem 40 (1992) 123.


Perhaps further reading: Cattini & Davies, “Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy,” Stain Technology 59 (1984) 291–304. Gluenz et al., “Structural asymmetry and discrete nucleic acid subdomains” in the Trypanosoma brucei kinetoplast,” Mol Microbiol 64 (2007) 1529–1539. Wolfgang Muss wij.muss@aon.at Tue Jun 6


LM: unknown microbe


I was looking at some pond water in a light microscope, and managed to capture this guy. Being a physicist, I have absolutely no idea what it might be, but I am sure that someone on this list will know what it is. Here is a video that I took of it: http://www.jkraſt .net/unknown-microbe.mp4 . Also, if anyone knows of a good internet source of information on how to identify the random little slimy bits in pond water, I would appreciate it. Justin Kraſt kraſt piano@gmail.comT u May 4


Contact the folks in Seth Tyler’s lab at University of Maine – they run a “global worming” website about metazoan microfauna, but they are mostly marine: http://globalworming.umaine-biology.net/ . Pennak’s “Freshwater Invertebrates of North America” is a good reference. Aſt er that, you need to get into the literature for each group, or google the group name, then click “images”. But. Squished by the coverslip and without a scale, it’s hard to tell. T e squishedness prevents the critter from exhibiting its true form. T ere are several sets of somethings … 2 near the anterior end, just behind the mouth (on the right), one about ½ way along the body, on top of the animal and hard to see (about where the gut makes the sharp bend), and another 2 spaced equally behind this, on the “top” of the animal as it sits. T is makes me think it’s a seriously uncomfortable tardigrade. Too fl attened by the coverslip to really show its true form. Common animals, but you need to put feet on your coverslips to give critters like this some room. Great dark-fi eld subjects as well as DIC. T rough in a green fi lter below your condenser, and use the 40× DIC annulus with the 10× objective, and you’ll have dark-fi eld (100× annulus and 20/40× objective will work on some microscopes). Given how poorly studied these groups are, it could be a new species. Philip Oshel oshel1pe@cmich.edu Fri May 5


Looks like it may be a freshwater Oligochaete, a type of Annelid worm (think earthworms). Couldn’t tell you what species it is though; that would take some careful keying as there are hundreds of them in North America (if that’s where you are!). If you look closely, it has what are called chaetae (bristles or hairs) in bunches on its body. T ere are quite a few keys to freshwater creatures. Here are just a few: Stroud Water Research


doi: 10.1017/S1551929517000761 www.microscopy-today.com • 2017 September


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