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in the oven. Let gelatin set completely. Mormyrids have a ginormous cerebellum that sits over the cerebrum and causes issues with the embedding medium getting to the rest of the brain. T is gelatin method worked for them, so it should work for chick brains. I hope. Phil Oshel oshel1pe@cmich.edu Tue Dec 5 T ere are some missing details. What is your fi xative or is this live? Are these expressing a fl uorescent protein or are you planning on immunolabeling? What instrument are you using to section and how are you attaching the sample to the sample holder. I’ve found that embryonic avian brain requires slow advance speed and high amplitude, whether live or fi xed. It is also very sensitive to knife angle. How are you attaching to the specimen holder? Not all cyanoacryates work with wet samples. I’ve had the best results orienting the tissue with the ventral surface facing the knife. T ere is a tendency for the sectioning shear force to push the brain apart when cut from the dorsal surface. As Phil mentions, surrounding membranes can cause shear forces that displace the sample. Or they drape around the razor’s edge and get dragged through the tissue. I’ve cut live embryonic brain down to 200 µm by gluing it to the holder and just using an agarose block to “backstop” the brain. Such a backstop can also help with any fragile tissue that does not off er much internal strength. In another project, embryonic avian brain was sectioned aſt er blocking the tissue to give a fl at base and gluing to the stub. T en low melting point agarose was ladled over it and allowed to harden. Another approach for small fl oppy tissues, like early embryonic brain or with spinal cord, is to cut the rounded end from a gelatin capsule or BEEM capsule and use the capsule as a mold for embedding with agarose. If embedded with the cap in place, remove the cap to expose the tissue. Or trim down the upper end to expose the tissue for slicing. Glen MacDonald glenmac@uw.edu Sun Dec 10


You can try Celloidin embedding media, with that you can section as thin as 15 µm I had used it for embedding Golgi stained human and mouse brain tissue. Here is the link for detailed description hope it helps you http://stainsfi le.info/StainsFile/prepare/process/celloidin. htm Arvind Singh Pundir arvind@nbrc.ac.in Sun Dec 10 Have you fi xed the tissue in paraformaldehyde? Karen Bentley karen_bentley@urmc.rochester.edu Sun Dec 10


Specimen Preparation: metal evaporation deposition units


Are there some suggestions about high vacuum evaporation deposition systems? I’m looking for something that would do high resolution deposition of Pt and Ir but does not have a large foot print like the Denton 502 units. T e Denton Desk IV TSC using Ir does not produce a fi ne enough coating for imaging at 25K×+. I’ve tried 15mT, 35% current and 60 seconds time but get clumps in my images using FEGSEM. Any ideas or suggestions, please? Gary Gaugler gary@ microtechnics.com Fri Dec 15


We recently purchased a Leica ACE 600 system equipped with sputter coater, carbon wire coater, and glow discharge options. We did not get a Pt target, but have Ir, Cr, Au, and AuPd. I think that it has a small foot print. It is easy to use. I use Ir. I found that the rate of deposition for a 1 nm coating was too fast, so I created a new recipe by decreasing the current by half to 40 mA. It takes about 30 sec, and I get good coatings. If you are getting clumps in your deposition, you should investigate more before investing in a new unit. Check your target for wear. Check that there is a good thermal connection. Check your gas supply for purity. You can also play around a bit with the pressure and power settings. Also, use as long a working distance as possible. Scott Walck s.walck@comcast.net Fri Dec 22


62


Fluorescence Microscopy: reducing autofl uorescence


I am trying to reduce the amount of autofl uorescence from my embryonic chick brains (E10 and E12), which are being imaged by light- sheet microscopy. Currently, I am drop-fi xing my heads in 4% PFA. T e embryos hearts are too delicate for us to perfuse them, so I was hoping to have another method to remove hemoglobin from the brain, and that should reduce my autofl uorescence. I have both GFP and Cholera toxin B-Alexa Fluor 555 that I am looking at with my imaging. Additionally, I am doing a BABB clearing method. Garrett Driscoll driscollg2@ winthrop.edu Fri Dec 15


Try 1% Sodium Borohydride in PBS for 10-15 min. Clancy and Cauller (1998). It won’t kill the fl uorescent proteins like some of the other treatments. Glen MacDonald glenmac@u.washington.edu Sun Dec 17 T ere are several other methods you can try that quench the remaining free aldehyde groups leſt aſt er glutaraldehyde fi xation (below). However, if you are not necessarily interested in ultrastructure, why not use paraformaldehyde? Aldehyde induced fl uorescence is usually much less of a problem with the PFA. You may also want to think about whether the fl uorescence you are seeing is indeed due to aldehydes - or if it could be due to some other cellular constituent like lysolipids. As an alternative, you may also want to consider using the longest wavelength your imaging and fl uorophore systems will allow. Try looking at the background fl uorescence above around 590 nm. It may be all you need to do.


For blocking free aldehydes remaining aſt er fi xation: • 1% (or 150 mM) glycine with 0.1% Tween in PBS. Wash well. Glycine binds to free aldehydes.


• 50 mM NH 4 Cl in PBS. Incubate for 15 min at room temperature. Wash well. (see Bendyan)


• 0.1% Na borohydride in PBS. Apply while fi zzing and incubate 3 × 10 min on ice. Wash well. T is reduces carbonyls.


• 0.15 M ethanolamine, pH 7.5. Incubate 30 min on ice. Wash well.


• Also from an earlier post by Scott Snyder ssnyder@ niaid.nih.gov : Probably the best way I have found of getting rid of aldehyde based fl uorescence is to get rid of the aldehydes. I oſt en do this by using the bifunctional crosslinking reagent dithio bis(succinimidyl propio- nate), or DSP for short. T is gives comparable structure retention in my hands and essentially no added fl uores- cence. A good reference for this is Safi eko-Mroczka and Bell, Journal of Histochemistry and Cytochemistry 44(6) (1996) 641–56.


Michael Cammer michael. cammer@med.nyu.edu Sun Dec 17 TEM:


oil diffusion pump (ODP) water message Today I tried to switch on our Philips CM10 TEM. Aſt er an hour the message “ODP water” appeared on the monitor. Reading the manual I fi gured out that the message was about high water temperature (60ºC) on the ODP cooling system. T ere is the S30 switch that checks that temperature. Our water cooling system seems to work fi ne (temperature about 15ºC). Any ideas how to fi x the problem? Panagiotis Berillis pveril@uth.gr Tue Dec 5


Check the thermal sensor on the ODP; it may need replacing. T is happened on my CM200. Nestor Zaluzec anl.nestor.zaluzec@gmail. com Tue Dec 5


www.microscopy-today.com • 2018 March


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