NetNotes


Edited by Thomas E. Phillips University of Missouri phillipst@missouri.edu


Selected postings to the Microscopy Listserver from May 1, 2011 to July 1, 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: polyvinylpyrrolidone in fi x In doing a literature search I came across a paper recommending


the use of polyvinylpyrrolidone (PVP) in glutaraldehyde fi xatives to help minimize changes due to osmotic pressure. In our case we would be using it on mouse intestinal tissue. Bohman and Maunsbach (1970) Eff ects on Tissue Fine Structure of Variations in Colloid Osmotic Pressure of Glutaraldehyde Fixatives. J. Ultrastructure Research 195–208. We oſt en do add sucrose to our fi x for osmotic control but I have never used PVP. I would appreciate comments from those who might have experience using it. Debby Sherman dsherman@purdue. edu Wed May 4 I used PVP many, many years ago when I was doing TEM on


the renal medulla, very hypertonic down there. How much the PVP helped versus how much adding lots of NaCl to the fi x to make it hypertonic helped is open to question. I got the recipe from Bohman’s work on the kidney which had very nice EMs. J. Ultrastruc. Res. 47:329–360, 1974. I actually met him some years later, nice guy and a good scientist. I have also done TEM of mouse duodenum and never had a problem getting good fi xation with 2.5% glutaraldehyde in buff er. I think I used 0.08 M cacodylate in those days but probably phosphate as well. I did not perfuse but just fl ushed the lumen with fi x from a small syringe with a 25 g needle. Final answer, it is probably not necessary but there is enough small intestine in a mouse to try adjacent pieces of tissue in fi x with and without PVP. Geoff McAuliff e mcauliff @umdnj.edu T u May 5


Specimen Preparation: shelf life of resins What are your ideas about the shelf life of embedding resins? We


have a bunch, some very old (a few back to 1983), and I don’t know what to do with them. Are they good to use or should they just go? I know I could mix up test sets, but I’m not sure I can spare the time. In addition, some of the things on the shelf I have never used. Some are old & open, some old and never opened. All kinds of things, DMAE, DMP-30, BDMA, Spurr’s components, Epon 812 substitutes, some Araldites, DBP, NMA, DDSA, Quetol, Maraglas, and Cardolite, LR White, the list goes on. All have been stored at room temperature in a glass door cabinet in the lab; most are in brown glass bottles. I’m ready to purge the lab of old, unused things, but don’t want to discard anything that would be useful for the future. Happy to share a complete list and give away what we don’t need. Jon Krupp jkrupp@deltacollege.edu T u May 5 In a slightly peripheral but related question, I was wondering


about the shelf life of Permount. We have a very old bottle in our lab, and it’s been opened some time in the past. We rarely do alcohol/ xylene series embedding, in fact, never have during the time I’ve been there. But if we were to, would an old bottle of Permount still be usable, or would the xylene base evaporated off too greatly to be usable? Peter Werner germpore@sonic.net T u May 5 Having attempted to use an old bottle of Permount, I’d suggest that you dispose of it now and get a new bottle when the time comes


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to coverslip again. If you can get the cap off you’d be lucky and the fl uid is most likely at least partially dehydrated and thick to the point that it will not fl ow properly under the coverslip. Patricia Stranen Connelly connellyps@nhlbi.nih.gov T u May 5 T e DMP-30 has a very short shelf life and the LR White should


have been in the refrigerator. Any old open bottles of NMA and DDSA and anything else that is supposed to be anhydrous need to be discarded. Unopened bottles of Epon 812 substitutes should be fi ne for years. My best suggestion would be to make a list with the lot numbers and send it to the suppliers and ask their opinions. Saving all the things in the world is not worth a failed embedding of an important experiment. Patricia Stranen Connelly connellyps@nhlbi. nih.gov T u May 5


Specimen Preparation: polystyrene beads for TEM A researcher wants to study the uptake of 200 nm polystyrene


(PS) beads using TEM of sectioned materials. I believe the beads will be dissolved in any solvent we use (including ethanol). I suggested other nanoparticles, but the researcher believes PS would be best for the work. I was hoping to use ethanol with the epoxy resin (to avoid more powerful organic solvents like acetone and polypropylene) but the manufacturer of the beads said any organic solvent would dissolve the beads. Anyone have experience using PS beads and embedding? John J. Bozzola bozzola@siu.edu Wed Jun 29 You might want to remember that some of the Epon substitutes


will also dissolve PS. I have used LX-112 from Ladd and it works well with PS. I assume that there are others that do also but EMbed-812 will not work with it for it melted my small polystyrene Petri dishes. Patricia Stranen Connelly connellyps@nhlbi.nih.gov Wed Jun 29 You could try Tokuyasu cryosectioning, I guess? Michal Jarnik


jarnikm@mail.nih.gov Wed Jun 29 While there is no doubt that acetone dissolves plastics, I


would be surprised if ethanol would do any harm to them. I would recommend bypassing the acetone step and directly embedding in ethanol/resin mixture aſt er dehydration. Just be sure to leave no trace of ethanol in the resin. In any case it is quite straightforward to test the resistance of the beads to ethanol or anything else: leave them in ethanol overnight and check by SEM. Stephane Nizets nizets2@ yahoo.com T u Jun 30 If you wish to test what solvents dissolve polystyrene try a low


cost styrofoam cup. It dissolves is seconds if you try to fi ll it with acetone. I tried it in chem lab decades ago, the lesson sticks. Since many alcoholic beverages are served in #6 PS cups, alcohol should be fi ne. Roseann Csencsits rcsencsits@lbl.gov T u Jun 30


Specimen Preparation: HMDS I wanted to test HMDS (hexamethyldisilazane) as an alternative to critical point drying for SEM of plant material and insects. 100%


doi:10.1017/S1551929511000915 www.microscopy-today.com • 2011 September


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